Nucleic acid-polypeptide compositions and uses thereof

ABSTRACT

Disclosed herein are compositions and pharmaceutical formulations that comprise a binding moiety conjugated to a polynucleic acid molecule and a polymer. Also described herein include methods for treating a cancer which utilize a composition or a pharmaceutical formulation comprising a binding moiety conjugated to a polynucleic acid molecule and a polymer.

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.15/476,849, filed Mar. 31, 2017, which in turn claims the benefit ofU.S. Provisional Application No. 62/316,919, filed Apr. 1, 2016, whichapplication is incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 11, 2018, isnamed 45532-707_303_SL.txt and is 615,705 bytes in size.

BACKGROUND OF THE DISCLOSURE

Gene suppression by RNA-induced gene silencing provides several levelsof control: transcription inactivation, small interfering RNA(siRNA)-induced mRNA degradation, and siRNA-induced transcriptionalattenuation. In some instances, RNA interference (RNAi) provides longlasting effect over multiple cell divisions. As such, RNAi represents aviable method useful for drug target validation, gene function analysis,pathway analysis, and disease therapeutics.

SUMMARY OF THE DISCLOSURE

Disclosed herein, in certain embodiments, are compositions andpharmaceutical formulations that comprise a binding moiety conjugated toa polynucleic acid molecule and a polymer. In some embodiments, alsodescribed herein include methods for treating a disease or condition(e.g., cancer) that utilize a composition or a pharmaceuticalformulation comprising a binding moiety conjugated to a polynucleic acidmolecule and a polymer.

Disclosed herein, in certain embodiments, is a molecule of Formula (I):A-X—B—Y—C   Formula I

-   -   wherein,        -   A is a binding moiety;        -   B is a polynucleotide;        -   C is a polymer;        -   X is a bond or first linker; and        -   Y is a bond or second linker; and    -   wherein the polynucleotide comprises at least one 2′ modified        nucleotide, at least one modified internucleotide linkage, or at        least one inverted abasic moiety.

In some embodiments, the at least one 2′ modified nucleotide comprises2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy,T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido(2′-O-NMA) modified nucleotide. In some embodiments, the at least one 2′modified nucleotide comprises locked nucleic acid (LNA) or ethylenenucleic acid (ENA). In some embodiments, the at least one modifiedinternucleotide linkage comprises a phosphorothioate linkage or aphosphorodithioate linkage. In some embodiments, the at least oneinverted abasic moiety is at at least one terminus.

In some embodiments, the polynucleotide comprises a single strand. Insome embodiments, the polynucleotide comprises two or more strands. Insome embodiments, the polynucleotide comprises a first polynucleotideand a second polynucleotide hybridized to the first polynucleotide toform a double-stranded polynucleic acid molecule. In some embodiments,the second polynucleotide comprises at least one modification.

In some embodiments, the first polynucleotide and the secondpolynucleotide are RNA molecules. In some embodiments, the firstpolynucleotide and the second polynucleotide are siRNA molecules.

In some embodiments, the first polynucleotide comprises a sequencehaving at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to SEQ ID NOs: 16-75, 452-1955,1956-1962, 1967-2002, 2013-2032, 2082-2109, or 2117. In someembodiments, the first polynucleotide consists of a sequence selectedfrom SEQ ID NOs: 16-75, 452-1955, 1956-1962, 1967-2002, 2013-2032,2082-2109, or 2117.

In some embodiments, the second polynucleotide comprises a sequencehaving at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to SEQ ID NOs: 16-75, 452-1955,1956-1962, 1967-2002, 2013-2032, 2082-2109, or 2117. In someembodiments, the second polynucleotide consists of a sequence selectedfrom SEQ ID NOs: 16-75, 452-1955, 1956-1962, 1967-2002, 2013-2032,2082-2109, or 2117.

In some embodiments, X and Y are independently a bond or a non-polymericlinker group. In some embodiments, X is a bond. In some embodiments, Xis a C₁-C₆ alkyl group. In some embodiments, Y is a C₁-C₆ alkyl group.In some embodiments, X is a homobifuctional linker or aheterobifunctional linker, optionally conjugated to a C₁-C₆ alkyl group.In some embodiments, Y is a homobifuctional linker or aheterobifunctional linker.

In some embodiments, the binding moiety is an antibody or bindingfragment thereof. In some embodiments, the antibody or binding fragmentthereof comprises a humanized antibody or binding fragment thereof,chimeric antibody or binding fragment thereof, monoclonal antibody orbinding fragment thereof, monovalent Fab′, divalent Fab2, single-chainvariable fragment (scFv), diabody, minibody, nanobody, single-domainantibody (sdAb), or camelid antibody or binding fragment thereof. Insome embodiments, the antibody or binding fragment thereof is ananti-EGFR antibody or binding fragment thereof.

In some embodiments, C is polyethylene glycol. In some embodiments, Chas a molecular weight of about 5000 Da.

In some embodiments, A-X is conjugated to the 5′ end of B and Y—C isconjugated to the 3′ end of B. In some embodiments, Y—C is conjugated tothe 5′ end of B and A-X is conjugated to the 3′ end of B. In someembodiments, A-X, Y—C or a combination thereof is conjugated to aninternucleotide linkage group.

In some embodiments, the molecule further comprises D. In someembodiments, D is conjugated to C or to A.

In some embodiments, D is conjugated to the molecule of Formula (I)according to Formula (II):(A-X—B—Y—C_(n))-L-D   Formula II

-   -   wherein,        -   A is a binding moiety;        -   B is a polynucleotide;        -   C is a polymer;        -   X is a bond or first linker;        -   Y is a bond or second linker;        -   L is a bond or third linker;        -   D is an endosomolytic moiety; and        -   n is an integer between 0 and 1; and    -   wherein the polynucleotide comprises at least one 2′ modified        nucleotide, at least one modified internucleotide linkage, or at        least one inverted abasic moiety; and D is conjugated anywhere        on A, B, or C.

In some embodiments, D is INF7 or melittin.

In some embodiments, D is an endosomolytic polymer.

In some embodiments, L is a C₁-C₆ alkyl group. In some embodiments, L isa homobifuctional linker or a heterobifunctional linker.

In some embodiments, the molecule further comprises at least a secondbinding moiety A. In some embodiments, the at least second bindingmoiety A is conjugated to A, to B, or to C. In some embodiments, the atleast second binding moiety A is cholesterol.

In some embodiments, the molecule further comprises at least anadditional polynucleotide B. In some embodiments, the at least anadditional polynucleotide B is conjugated to A, to B, or to C.

In some embodiments, the molecule further comprises at least anadditional polymer C. In some embodiments, the at least an additionalpolymer C is conjugated to A, to B, or to C.

Disclosed herein, in certain embodiments, is a molecule of Formula (I):A-X—B—Y—C (Formula I), wherein A is an antibody or its binding fragmentsthereof; B is a polynucleotide; C is a polymer; X is a bond or firstnon-polymeric linker; and Y is a bond or second linker; wherein thepolynucleotide comprises at least one 2′ modified nucleotide, at leastone modified internucleotide linkage, or at least one inverted abasicmoiety; and wherein A and C are not attached to B at the same terminus.In some embodiments, the at least one 2′ modified nucleotide comprises2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy,T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido(2′-O-NMA) modified nucleotide. In some embodiments, the at least one 2′modified nucleotide comprises locked nucleic acid (LNA) or ethylenenucleic acid (ENA). In some embodiments, the at least one modifiedinternucleotide linkage comprises a phosphorothioate linkage or aphosphorodithioate linkage. In some embodiments, the at least oneinverted abasic moiety is at at least one terminus. In some embodiments,the polynucleotide comprises a single strand. In some embodiments, thepolynucleotide comprises a first polynucleotide and a secondpolynucleotide hybridized to the first polynucleotide to form adouble-stranded polynucleic acid molecule. In some embodiments, thesecond polynucleotide comprises at least one modification. In someembodiments, the first polynucleotide and the second polynucleotide areRNA molecules. In some embodiments, the first polynucleotide comprises asequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to SEQ ID NOs: 16-75, 452-1955, 1956-1962, 1967-2002,2013-2032, 2082-2109, or 2117. In some embodiments, the secondpolynucleotide comprises a sequence having at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 16-75,452-1955, 1956-1962, 1967-2002, 2013-2032, 2082-2109, or 2117. In someembodiments, Y is a non-polymeric linker group. In some embodiments, Xis a bond. In some embodiments, X is a C₁-C₆ alkyl group. In someembodiments, Y is a C₁-C₆ alkyl group. In some embodiments, X is ahomobifuctional linker or a heterobifunctional linker, optionallyconjugated to a C₁-C₆ alkyl group. In some embodiments, Y is ahomobifuctional linker or a heterobifunctional linker. In someembodiments, the antibody or binding fragment thereof comprises ahumanized antibody or binding fragment thereof, chimeric antibody orbinding fragment thereof, monoclonal antibody or binding fragmentthereof, monovalent Fab′, divalent Fab2, single-chain variable fragment(scFv), diabody, minibody, nanobody, single-domain antibody (sdAb), orcamelid antibody or binding fragment thereof. In some embodiments, C ispolyethylene glycol. In some embodiments, C has a molecular weight ofabout 1000 Da, 2000 Da, or 5000 Da. In some embodiments, A-X isconjugated to the 5′ end of B and Y—C is conjugated to the 3′ end of B.In some embodiments, Y—C is conjugated to the 5′ end of B and A-X isconjugated to the 3′ end of B. In some embodiments, the molecule furthercomprises D. In some embodiments, D is conjugated to C or to A. In someembodiments, D is conjugated to the molecule of Formula (I) according toFormula (II): (A-X—B—Y—C_(c))-L-D (Formula H), wherein A is an antibodyor its binding fragments thereof; B is a polynucleotide; C is a polymer;X is a bond or first non-polymeric linker; Y is a bond or second linker;L is a bond or third linker; D is an endosomolytic moiety; and c is aninteger between 0 and 1; wherein the polynucleotide comprises at leastone 2′ modified nucleotide, at least one modified internucleotidelinkage, or at least one inverted abasic moiety; wherein A and C are notattached to B at the same terminus; and wherein D is conjugated anywhereon A or C or to a terminus of B. In some embodiments, D is INF7 ormelittin. In some embodiments, D is an endosomolytic polymer. In someembodiments, L is a C₁-C₆ alkyl group. In some embodiments, L is ahomobifuctional linker or a heterobifunctional linker. In someembodiments, the molecule further comprises at least a second bindingmoiety. In some embodiments, the at least second binding moiety isconjugated to A, to B, or to C. In some embodiments, the at least secondbinding moiety is cholesterol. In some embodiments, the molecule furthercomprises at least an additional polynucleotide B. In some embodiments,the at least an additional polynucleotide B is conjugated to A, to B, orto C. In some embodiments, the molecule further comprises at least anadditional polymer C. In some embodiments, the at least an additionalpolymer C is conjugated to A, to B, or to C.

Disclosed herein, in certain embodiments, is a pharmaceuticalcomposition comprising a molecule described above, and apharmaceutically acceptable excipient. In some embodiments, thepharmaceutical composition is formulated as a nanoparticle formulation.In some embodiments, the pharmaceutical composition is formulated forparenteral, oral, intranasal, buccal, rectal, or transdermaladministration.

Disclosed herein, in certain embodiments, is a method of treating adisease or disorder in a patient in need thereof, comprisingadministering to the patient a composition comprising a moleculedescribed above. In some embodiments, the disease or disorder is acancer. In some embodiments, the cancer is a solid tumor. In someembodiments, the cancer is a hematologic malignancy. In someembodiments, the cancer comprises a KRAS-associated, an EGFR-associated,an AR-associated cancer, a β-catenin associated cancer, aPIK3C-associated cancer, or a MYC-associated cancer. In someembodiments, the cancer comprises bladder cancer, breast cancer,colorectal cancer, endometrial cancer, esophageal cancer, glioblastomamultiforme, head and neck cancer, kidney cancer, lung cancer, ovariancancer, pancreatic cancer, prostate cancer, or thyroid cancer. In someembodiments, the cancer comprises acute myeloid leukemia, CLL, DLBCL, ormultiple myeloma. In some embodiments, the method is an immuno-oncologytherapy.

Disclosed herein, in certain embodiments, is a method of inhibiting theexpression of a target gene in a primary cell of a patient, comprisingadministering a molecule described above to the primary cell. In someembodiments, the method is an in vivo method. In some embodiments, thepatient is a human.

Disclosed herein, in certain embodiments, is an immuno-oncology therapycomprising a molecule described above for the treatment of a disease ordisorder in a patient in need thereof.

Disclosed herein, in certain embodiments, is a kit comprising a moleculedescribed above.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of the disclosure are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present disclosure will be obtained by reference tothe following detailed description that sets forth illustrativeembodiments, in which the principles of the disclosure are utilized, andthe accompanying drawings below.

FIGS. 1A-FIG. 1V illustrate cartoon representations of moleculesdescribed herein.

FIG. 2 illustrates a structure of cholesterol conjugate passengerstrand.

FIG. 3 shows an INF7 peptide sequence (SEQ ID NO: 2055) describedherein.

FIG. 4 shows a melittin peptide sequence (SEQ ID NO: 2060) describedherein.

FIG. 5 illustrates an analytical HPLC of EGFR antibody-PEG20 kDa-EGFR.

FIG. 6 illustrates a SDS-PAGE analysis of EGFR antibody-PEG20 kDa-EGFRconjugate.

FIG. 7 illustrates an analytical chromatogram of EGFR antibody-PEG10kDa-EGFR siRNA.

FIG. 8 shows an analytical chromatogram of EGFR antibody-PEG5 kDa-EGFRsiRNA.

FIG. 9 shows a SDS PAGE analysis of EGFR antibody-PEG10 kDa-EGFR siRNAand EGFR antibody-PEG5 kDa-EGFR siRNA conjugates.

FIG. 10 illustrates the overlay of EGFR antibody-PEG1 kDa-EGFR siRNAconjugates with siRNA loading of 1, 2 and 3.

FIG. 11 shows a HPLC chromatogram of EGFR antibody-KRAS-PEG5 kDa.

FIG. 12 shows a HPLC chromatogram of Panitumumab-KRAS-PEG5 kDa.

FIG. 13 shows a HPLC chromatogram of EGFR antibody-S—S-siRNA-PEG5 kDa(DAR=1).

FIG. 14 shows a HPLC chromatogram of EGFR antibody-PEG24-Melittin(loading=˜1).

FIG. 15 illustrates a HPLC chromatogram of EGFR antibody-Melittin(n=˜0.1).

FIG. 16 illustrates a mass spectrum of EGFR antibody-Melittin (n=1).

FIG. 17 shows a HIC chromatogram of EGFR antibody-PEG1 kDa-INF7 (Peptideloading=˜1).

FIG. 18 shows a HPLC chromatogram of EGFR antibody-INF7 (PeptideLoading=˜1).

FIG. 19 shows INF7-PEG1 kDa-(EGFR antibody-KRAS-PEG5 kDa).

FIG. 20 illustrates Melittin-PEG1 kDa-(EGFR antibody-KRAS-PEG5 kDa).

FIG. 21 illustrates plasma concentration-time profiles out to 96 hpost-dose with the siRNA concentration expressed as a percent ofinjected dose (% ID).

FIG. 22 shows plasma concentration-time profiles out to 96 h post-dosewith the siRNA concentration expressed as a percent of injected dose (%ID).

FIG. 23 shows plasma concentration-time profiles out to 96 h post-dosewith the siRNA concentration expressed as a percent of injected dose (%ID).

FIG. 24 illustrates plasma concentration-time profiles out to 96 hpost-dose with the siRNA concentration expressed as a percent ofinjected dose (% ID).

FIG. 25 illustrates plasma concentration-time profiles out to 24 hpost-dose with the siRNA concentration expressed as a percent ofinjected dose (% ID).

FIG. 26A and FIG. 26B illustrate tissue concentration-time profiles intumor or normal livers of mice. FIG. 26A shows tissue concentration-timeprofiles out to 168 h post-dose measured in s.c. flank H358 tumors in amice model. FIG. 26B shows tissue concentration-time profiles out to 168h post-dose measured in normal livers of mice.

FIG. 27 shows tissue concentration-time profiles out to 168 h post-dosemeasured in s.c. flank H358 tumors and normal livers of mice.

FIG. 28 illustrates tissue concentration-time profiles out to 168 hpost-dose measured in s.c. flank H358 tumors and normal livers of mice.

FIG. 29 illustrates tissue concentration-time profiles out to 168 hpost-dose measured in s.c. flank H358 tumors and normal livers of mice.

FIG. 30 shows tissue concentration-time profiles out to 168 h post-dosemeasured in s.c. flank H358 tumors and normal livers of mice.

FIG. 31A and FIG. 31B illustrate siRNA-mediated mRNA knockdown of humanKRAS in human s.c. flank H358 tumors (FIG. 31A) or mouse KRAS in normalmouse liver (FIG. 31B).

FIG. 32 illustrates siRNA-mediated mRNA knockdown of human EGFR in humans.c. flank H358 tumors.

FIG. 33 illustrates siRNA-mediated mRNA knockdown of human KRAS in humans.c. flank H358 tumors.

FIG. 34 illustrates siRNA-mediated mRNA knockdown of human EGFR in humans.c. flank H358 tumors.

FIG. 35 shows siRNA-mediated mRNA knockdown of mouse KRAS in mouseliver.

FIG. 36 illustrates plasma concentration-time profiles out to 96 hpost-dose with the siRNA concentration expressed as a percent ofinjected dose (% ID).

FIG. 37 illustrates tissue concentration-time profiles out to 144 hpost-dose measured in liver, kidneys, and lungs of wild-type CD-1 mice.

FIG. 38A and FIG. 38B illustrate tissue concentration-time profiles outto 144 h post-dose measured in human s.c. flank H358 tumors forchol-KRAS mixed with either chol-INF7 peptide (FIG. 38A) orchol-melittin peptide (FIG. 38B).

FIG. 39A and FIG. 39B illustrate tissue concentration-time profiles outto 144 h post-dose measured in mouse liver for chol-KRAS mixed witheither chol-INF7 peptide (FIG. 39A) or chol-melittin peptide (FIG. 39B).

FIG. 40A and FIG. 40B illustrate tissue concentration-time profiles outto 144 h post-dose measured in mouse kidneys for chol-KRAS mixed witheither chol-INF7 peptide (FIG. 40A) or chol-melittin peptide (FIG. 40B).

FIG. 41A and FIG. 41B illustrate tissue concentration-time profiles outto 144 h post-dose measured in mouse lungs for chol-KRAS mixed witheither chol-INF7 peptide (FIG. 41A) or chol-melittin peptide (FIG. 41B).

FIG. 42 illustrates siRNA-mediated mRNA knockdown of mouse KRAS in mouseliver.

FIG. 43A and FIG. 43B illustrate tissue concentration-time profiles outto 96 h post-dose measured in human s.c. flank H358 tumors (FIG. 43A) ormouse liver (FIG. 43B).

FIG. 44A and FIG. 44B show tissue concentration-time profiles out to 96h post-dose measured in mouse kidneys (FIG. 44A) or mouse lungs (FIG.44B).

FIG. 45 shows siRNA-mediated mRNA knockdown of mouse KRAS in human s.c.flank H358 tumors.

FIG. 46 shows tissue concentrations of siRNA at 96 h post-dose measuredin human s.c. flank H358 tumors and mouse liver, kidneys, and lungs.

FIG. 47A and FIG. 47B show siRNA-mediated mRNA knockdown in human s.c.flank H358 tumors of EGFR (FIG. 47A) or KRAS (FIG. 47B).

FIG. 48 shows siRNA-mediated mRNA knockdown of human CTNNB1 in Hep3Borthotopic liver tumors.

FIG. 49 shows human alpha-Fetoprotein in serum from mice with Hep3Borthotopic liver tumors.

FIG. 50A shows siRNA-mediated mRNA knockdown of human EGFR in LNCaPtumor.

FIG. 50B shows siRNA concentration in tumor or liver tissues at 96 hourpost-dose.

FIG. 51A illustrates siRNA-mediated mRNA knockdown of human EGFR inLNCaP tumor at 96 hour.

FIG. 51B shows siRNA concentration in tumor or liver tissues at 96 hourpost-dose.

FIG. 52 shows plasma siRNA concentration of exemplary moleculesdescribed herein.

FIG. 53A illustrates siRNA concentration of exemplary moleculesdescribed herein in HCC827 tumor or liver tissue.

FIG. 53B shows EGFR EGFR mRNA expression level of exemplary moleculesdescribed herein.

FIG. 54 illustrates exemplary As and Bs to generate moleculesencompassed by Formula (I).

FIG. 55 illustrates EGFR mRNA expression level of exemplary moleculesdescribed herein.

FIG. 56A illustrates siRNA concentration of exemplary moleculesdescribed herein in HCC827 tumor or liver tissue.

FIG. 56B shows EGFR mRNA expression level of exemplary moleculesdescribed herein.

FIG. 57A-FIG. 57B illustrate siRNA concentration of exemplary moleculesdescribed herein in liver (FIG. 57A) and tumor (FIG. 57B).

FIG. 57C shows KRAS mRNA expression level of exemplary moleculesdescribed herein.

FIG. 58A illustrates plasma siRNA concentration of exemplary moleculesdescribed herein.

FIG. 58B shows plasma antibody concentration of exemplary moleculesdescribed herein.

FIG. 59A illustrates siRNA concentration of exemplary moleculesdescribed herein in tumor or liver tissue.

FIG. 59B shows mRNA expression level of exemplary molecules describedherein in Hep3B tumor.

FIG. 60 shows CTNNB1 mRNA expression level of an exemplary moleculedescribed herein in liver.

FIG. 61 shows KRAS mRNA expression level of an exemplary moleculedescribed herein in liver.

FIG. 62 illustrates plasma siRNA or monoclonal antibody (mAb)concentration of exemplary molecules described herein.

FIG. 63A illustrates siRNA concentration of exemplary moleculesdescribed herein in tumor or liver tissue.

FIG. 63B shows EGFR mRNA expression level of exemplary moleculesdescribed herein in LNCaP tumor.

FIG. 64A-FIG. 64E illustrate HPRT mRNA expression level in heart (FIG.64A), HPRT mRNA expression level in gastrointestinal tissue (FIG. 64B),HPRT mRNA expression level in liver (FIG. 64C), HPRT mRNA expressionlevel in lung (FIG. 64D), and siRNA concentration in tissue (FIG. 64E)of exemplary molecules described herein.

FIG. 65A-FIG. 65E illustrate mRNA expression level in heart (FIG. 65A),mRNA expression level in gastrointestinal tissue (FIG. 65B), mRNAexpression level in liver (FIG. 65C), mRNA expression level in lung(FIG. 65D), and siRNA concentration in tissue (FIG. 65E) of exemplarymolecules described herein.

FIG. 66A-FIG. 66D illustrate siRNA concentration in heart (FIG. 66A),mRNA expression level in heart (FIG. 66B), mRNA expression level ingastrointestinal tissue (FIG. 66C), and siRNA concentration in muscle(FIG. 66D).

FIG. 67A illustrate mRNA expression level of exemplary moleculesdescribed herein.

FIG. 67B shows siRNA concentration of exemplary molecules describedherein in tumor or liver tissues.

FIG. 68A-FIG. 68B illustrate anti-B cell antibody-siRNA conjugates whichactivate primary mouse B cells. FIG. 68A illustrates an anti-B cellFab-siRNA conjugate. FIG. 68B shows an anti-B cell monoclonalantibody-siRNA conjugate.

FIG. 69A illustrates plasma siRNA concentration of exemplary moleculesdescribed herein.

FIG. 69B shows antibody zalutumumab concentration of exemplary moleculesdescribed herein in the plasma at a 5 mg/kg dose.

FIG. 70A shows mRNA expression level of exemplary molecules describedherein.

FIG. 70B shows siRNA concentration of exemplary molecules describedherein in tumor or liver tissues.

FIG. 70C shows plasma siRNA concentration of exemplary moleculesdescribed herein.

FIG. 71A illustrates siRNA concentration of exemplary moleculesdescribed herein in LNCaP tomor.

FIG. 71B-FIG. 71C illustrate mRNA expression level of exemplarymolecules described herein in LNCaP tomor.

FIG. 72A illustrates siRNA concentration of exemplary moleculesdescribed herein in tissue.

FIG. 72B shows mRNA expression level of exemplary molecules describedherein in HCC827 tumors at 96 h post-treatment.

FIG. 73A illustrates siRNA concentration of exemplary moleculesdescribed herein in the plasma at a 0.5 mg/kg dose.

FIG. 73B shows antibody zalutumumab concentration of exemplary moleculesdescribed herein in the plasma at a 5 mg/kg dose.

FIG. 74 illustrates plasma clearance of exemplary molecules encompassedby Formula (I) which contains different linkers.

FIG. 75A illustrates the mRNA expression level of exemplary moleculesdescribed herein in HCC827 tumor at a 0.5 mg/kg dose.

FIG. 75B-FIG. 75D illustrate siRNA concentration in tumor (FIG. 75B),liver (FIG. 75C), and plasma (FIG. 75D).

FIG. 76A-FIG. 76D illustrate mRNA expression levels of exemplarymolecules described herein targeting HPRT. FIG. 76A shows the mRNAexpression level in heart. FIG. 76B shows the mRNA expression level inmuscle. FIG. 76C shows the mRNA expression level in liver. FIG. 76Dshows the mRNA expression level in lung.

FIG. 77A-FIG. 77D illustrate siRNA concentrations of exemplary moleculesencompassed by Formula (I) in muscle (FIG. 77A), heart (FIG. 77B), liver(FIG. 77C), and lung (FIG. 77D).

FIG. 78A-FIG. 78D illustrate mRNA expression levels of exemplarymolecules encompassed by Formula (I) in heart (FIG. 78A),gastrointestinal tissue (FIG. 78B), liver (FIG. 78C), and lung (FIG.78D) at 96 h post-treatment.

FIG. 79 illustrates plasma siRNA concentration of exemplary moleculesencompassed by Formula (I).

FIG. 80A shows mRNA expression level of exemplary molecules encompassedby Formula (I) in LNCaP tumor at 96 h post-treatment.

FIG. 80B shows siRNA concentration of exemplary molecules encompassed byFormula (I) in LNCaP tumor, liver, kidney, lung, and spleen tissuesamples.

FIG. 81A shows mRNA expression level of exemplary molecules encompassedby Formula (I) in HCC827 tumor at 96 h post-treatment.

FIG. 81B illustrates siRNA concentrations of exemplary moleculesencompassed by Formula (I) in tumor, liver, kidney, lung, and spleentissue samples.

FIG. 82 illustrates plasma siRNA concentration of exemplary moleculesencompassed by Formula (I).

FIG. 83 illustrates plasma siRNA concentration of exemplary moleculesencompassed by Formula (I).

FIG. 84 illustrates mRNA expression levels of exemplary moleculesencompassed by Formula (I) in HCC827 tumor at 96 h post treatment.

FIG. 85 illustrates siRNA concentration in HCC827 tumor or liver tissuesat 96 hour post-dose.

FIG. 86 illustrates the relative mRNA expression levels of exemplarymolecules encompassed by Formula (I) in mouse splenic B cells 48 h posttreatment. Each exemplary molecule is further denoted with a number.

FIG. 87 illustrates stability of exemplary molecules encompassed byFormula (I) (or ASCs) in mouse plasma.

FIG. 88 illustrates Conjugation scheme 1.

FIG. 89 illustrates Conjugation scheme 2.

FIG. 90 illustrates Conjugation scheme 3.

FIG. 91 illustrates Conjugation scheme 4.

FIG. 92 illustrates Conjugation scheme 5.

FIG. 93 illustrates Conjugation scheme 6.

FIG. 94 illustrates Conjugation scheme 7.

FIG. 95 illustrates Conjugation scheme 8.

FIG. 96 illustrates Conjugation scheme 9.

FIG. 97 illustrates Conjugation scheme 10.

FIG. 98 illustrates Conjugation scheme 11.

FIG. 99 illustrates Conjugation scheme 12.

FIG. 100 illustrates Conjugation scheme 13.

FIG. 101 illustrates Conjugation scheme 14.

FIG. 102 illustrates Conjugation scheme 15.

FIG. 103 illustrates Conjugation scheme 16.

FIG. 104 illustrates a representative structure of siRNA with C6-NH₂conjugation handle at the 5′ end and C6-SH at 3′end of the passengerstrand.

FIG. 105 illustrates Antibody-Lys-SMCC-S-3′-Passenger strand.

FIG. 106 illustrates Antibody-Cys-SMCC-3′-Passenger strand.

FIG. 107 illustrates Antibody-Lys-SMCC-S-5′-passenger strand.

FIG. 108 illustrates Antibody-Cys-SMCC-5′-passenger strand.

FIG. 109 illustrates Antibody-Lys-PEG-5′-passenger strand.

FIG. 110 illustrates Antibody-Lys-PEG-5′-passenger strand.

FIG. 111 illustrates Antibody-Cys-PEG-5′-passenger strand withoutinverted abasic at 5′ end.

DETAILED DESCRIPTION OF THE DISCLOSURE

Nucleic acid (e.g., RNAi) therapy is a targeted therapy with highselectivity and specificity. However, in some instances, nucleic acidtherapy is also hindered by poor intracellular uptake, limited bloodstability and non-specific immune stimulation. To address these issues,various modifications of the nucleic acid composition are explored, suchas for example, novel linkers for better stabilizing and/or lowertoxicity, optimization of binding moiety for increased targetspecificity and/or target delivery, and nucleic acid polymermodifications for increased stability and/or reduced off-target effect.

In some embodiments, the arrangement or order of the differentcomponents that make-up the nucleic acid composition further effectsintracellular uptake, stability, toxicity, efficacy, and/or non-specificimmune stimulation. For example, if the nucleic acid component includesa binding moiety, a polymer, and a polynucleic acid molecule (orpolynucleotide), the order or arrangement of the binding moiety, thepolymer, and/or the polynucleic acid molecule (or polynucleotide) (e.g.,binding moiety-polynucleic acid molecule-polymer, bindingmoiety-polymer-polynucleic acid molecule, or polymer-bindingmoiety-polynucleic acid molecule) further effects intracellular uptake,stability, toxicity, efficacy, and/or non-specific immune stimulation.

In some embodiments, described herein include a molecule thosearrangement of the nucleic acid components effects intracellular uptake,stability, toxicity, efficacy, and/or non-specific immune stimulation.In some instances, the molecule comprises a binding moiety conjugated toa polynucleic acid molecule and a polymer. In some embodiments, themolecule comprises a molecule according to Formula (I): A-X—B—Y—C; inwhich A is a binding moiety, B is a polynucleotide, C is a polymer, X isa bond or first linker, and Y is a bond or second linker. In someinstances, the polynucleotide comprises at least one 2′ modifiednucleotide, at least one modified internucleotide linkage, or at leastone inverted abasic moiety. In some instances, the molecule of Formula(I) further comprises D, an endosomolytic moiety.

In some embodiments, a molecule comprising a binding moiety conjugatedto a polynucleic acid molecule and a polymer arranged as describedherein enhances intracellular uptake, stability, and/or efficacy. Insome instances, a molecule comprising a binding moiety conjugated to apolynucleic acid molecule and a polymer arranged as described hereinreduces toxicity and/or non-specific immune stimulation. In some cases,the molecule comprises a molecule according to Formula (I): A-X—B—Y—C;in which A is a binding moiety, B is a polynucleotide, C is a polymer, Xis a bond or first linker, and Y is a bond or second linker. In someinstances, the polynucleotide comprises at least one 2′ modifiednucleotide, at least one modified internucleotide linkage, or at leastone inverted abasic moiety. In some instances, the molecule of Formula(I) further comprises D, an endosomolytic moiety.

In some embodiments, a molecule described herein is further used totreat a disease or disorder. In some instances, a molecule for thetreatment of a disease or disorder is a molecule according to Formula(I): A-X—B—Y—C; in which A is a binding moiety, B is a polynucleotide, Cis a polymer, X is a bond or first linker, and Y is a bond or secondlinker. In some instances, the polynucleotide comprises at least one 2′modified nucleotide, at least one modified internucleotide linkage, orat least one inverted abasic moiety. In some instances, the molecule ofFormula (I) further comprises D, an endosomolytic moiety.

In some embodiments, a molecule described herein is also used forinhibiting the expression of a target gene in a primary cell of apatient in need thereof. In such instances, a molecule for such use is amolecule according to Formula (I): A-X—B—Y—C; in which A is a bindingmoiety, B is a polynucleotide, C is a polymer, X is a bond or firstlinker, and Y is a bond or second linker. In some instances, thepolynucleotide comprises at least one 2′ modified nucleotide, at leastone modified internucleotide linkage, or at least one inverted abasicmoiety. In some instances, the molecule of Formula (I) further comprisesD, an endosomolytic moiety.

In some embodiments, a molecule described herein is additionally used asan immuno-oncology therapy for the treatment of a disease or disorder.In some instance, the molecule is a molecule according to Formula (I):A-X—B—Y—C; in which A is a binding moiety, B is a polynucleotide, C is apolymer, X is a bond or first linker, and Y is a bond or second linker.In some instances, the polynucleotide comprises at least one 2′ modifiednucleotide, at least one modified internucleotide linkage, or at leastone inverted abasic moiety. In some instances, the molecule of Formula(I) further comprises D, an endosomolytic moiety.

In additional embodiments, described herein include a kit, whichcomprises one or more of the molecules described herein.

Therapeutic Molecule Platform

In some embodiments, a molecule (e.g., a therapeutic molecule) describedherein comprises a binding moiety conjugated to a polynucleic acidmolecule and a polymer. In some embodiments, a molecule (e.g., atherapeutic molecule) comprises a molecule according to Formula (I):A-X—B—Y—C   Formula Iwherein,

A is a binding moiety;

B is a polynucleotide;

C is a polymer;

X is a bond or first linker; and

Y is a bond or second linker; and

wherein the polynucleotide comprises at least one 2′ modifiednucleotide, at least one modified internucleotide linkage, or at leastone inverted abasic moiety.

In some instances, the molecule of Formula (I) further comprises D, anendosomolytic moiety.

In some embodiments, at least one A and/or at least one C are conjugatedto the 5′ terminus of B, the 3′ terminus of B, an internal site on B, orin any combinations thereof. In some instances, at least one A isconjugated at one terminus of B while at least one C is conjugated atthe opposite terminus of B. In some instances, at least one of A isconjugated at one terminus of B while at least one of C is conjugated atan internal site on B.

In some cases, A and C are not conjugated or attached to B at the sameterminus. In some cases, A is attached or conjugated to B at a firstterminus of B. In some cases, C is attached or conjugated to B at asecond terminus of B, and the second terminus of B is different than thefirst terminus. In some cases, A is attached or conjugated to B at the5′ terminus of B, and C is attached or conjugated to B at the 3′terminus of B. In other cases, A is attached or conjugated to B at the3′ terminus of B, and C is attached or conjugated to B at the 5′terminus of B.

In some embodiments, A is an antibody or binding fragment thereof. Insome cases, C is a polymer. In some cases, A and C are not conjugated orattached to B at the same terminus. In some cases, A is attached orconjugated to B at a first terminus of B. In some cases, C is attachedor conjugated to B at a second terminus of B, and the second terminus ofB is different than the first terminus. In some cases, A is attached orconjugated to B at the 5′ terminus of B, and C is attached or conjugatedto B at the 3′ terminus of B. In other cases, A is attached orconjugated to B at the 3′ terminus of B, and C is attached or conjugatedto B at the 5′ terminus of B. In some cases, X which connects A to B isa bond or a non-polymeric linker. In some cases, X is a non-peptidelinker (or a linker that does not comprise an amino acid residue). Insome cases, Y which connects B to C is a bond or a second linker. Insome instances, X connects A to the 5′ terminus of B, and Y connects Cto the 3′ terminus of B. In other instances, X connects A to the 3′terminus of B, and Y connects C to the 5′ terminus of B.

In some embodiments, X—B is conjugated or attached to the N-terminus,C-terminus, a constant region, a hinge region, or a Fc region of A. Insome instances, X—B is conjugated or attached to the N-terminus of A. Insome instances, X—B is conjugated or attached to the C-terminus of A. Insome instances, X—B is conjugated or attached to a hinge region of A. Insome instances, X—B is conjugated or attached to a constant region of A.In some instances, X—B is conjugated or attached to the Fc region of A.

In some instances, at least one B and/or at least one C, and optionallyat least one D are conjugated to a first A. In some instances, the atleast one B is conjugated at a terminus (e.g., a 5′ terminus or a 3′terminus) to the first A or are conjugated via an internal site to thefirst A. In some cases, the at least one C is conjugated either directlyto the first A or indirectly via the two or more Bs. If indirectly viathe two or more Bs, the two or more Cs are conjugated either at the sameterminus as the first A on B, at opposing terminus from the first A, orindependently at an internal site. In some instances, at least oneadditional A is further conjugated to the first A, to B, or to C. Inadditional instances, the at least one D is optionally conjugated eitherdirectly or indirectly to the first A, to the at least one B, or to theat least one C. If directly to the first A, the at least one D is alsooptionally conjugated to the at least one B to form a A-D-B conjugate oris optionally conjugated to the at least one B and the at least one C toform a A-D-B—C conjugate. In some cases, the at least one additional Ais different than the first A.

In some cases, two or more Bs and/or two or more Cs are conjugated to afirst A. In some instances, the two or more Bs are conjugated at aterminus (e.g., a 5′ terminus or a 3′ terminus) to the first A or areconjugated via an internal site to the first A. In some instances, thetwo or more Cs are conjugated either directly to the first A orindirectly via the two or more Bs. If indirectly via the two or more Bs,the two or more Cs are conjugated either at the same terminus as thefirst A on B, at opposing terminus from the first A, or independently atan internal site. In some instances, at least one additional A isfurther conjugated to the first A, to two or more Bs, or to two or moreCs. In additional instances, at least one D is optionally conjugatedeither directly or indirectly to the first A, to the two or more Bs, orto the two or more Cs. If indirectly to the first A, the at least one Dis conjugated to the first A through the two or more Bs, through the twoor more Cs, through a B—C orientation to form a A-B—C-D type conjugate,or through a C—B orientation to form a A-C—B-D type conjugate. In somecases, the at least one additional A is different than the first A. Insome cases, the two or more Bs are different. In other cases, the two ormore Bs are the same. In some instances, the two or more Cs aredifferent. In other instances, the two or more Cs are the same. Inadditional instances, the two or more Ds are different. In additionalinstances, the two or more Ds are the same.

In other cases, two or more Bs and/or two or more Ds, optionally two ormore Cs are conjugated to a first A. In some instances, the two or moreBs are conjugated at a terminus (e.g., a 5′ terminus or a 3′ terminus)to the first A or are conjugated via an internal site to the first A. Insome instances, the two or more Ds are conjugated either directly to thefirst A or indirectly via the two or more Bs. If indirectly via the twoor more Bs, the two or more Ds are conjugated either at the sameterminus as the first A on B, at opposing terminus from the first A, orindependently at an internal site. In some instances, at least oneadditional A is further conjugated to the first A, to the two or moreBs, or to the two or more Ds. In additional instances, the two or moreCs are optionally conjugated either directly or indirectly to the firstA, to the two or more Bs, or to the two or more Ds. In some cases, theat least one additional A is different than the first A. In some cases,the two or more Bs are different. In other cases, the two or more Bs arethe same. In some instances, the two or more Cs are different. In otherinstances, the two or more Cs are the same. In additional instances, thetwo or more Ds are different. In additional instances, the two or moreDs are the same.

In some embodiments, a molecule (e.g., a therapeutic molecule) describedherein comprises a molecule according to Formula (II):(A-X—B—Y—C_(c))-L-D   Formula IIwherein,

A is a binding moiety;

B is a polynucleotide;

C is a polymer;

X is a bond or first linker;

Y is a bond or second linker;

L is a bond or third linker;

D is an endosomolytic moiety; and

c is an integer between 0 and 1; and

wherein the polynucleotide comprises at least one 2′ modifiednucleotide, at least one modified internucleotide linkage, or at leastone inverted abasic moiety; and D is conjugated anywhere on A, B, or C.

In some embodiments, a molecule (e.g., a therapeutic molecule) describedherein comprises a molecule according to Formula (III):A_(a)-X—B_(b)—Y—C_(c)-L-D_(n)   Formula IIIwherein,

A is a binding moiety;

B is a polynucleotide;

C is a polymer;

D is an endosomolytic moiety;

X is a bond or first linker;

Y is a bond or second linker;

L is a bond or third linker;

a and b are independently an integer between 1-3;

c is an integer between 0 and 3; and

n is an integer between 0 and 10; and

wherein the polynucleotide comprises at least one 2′ modifiednucleotide, at least one modified internucleotide linkage, or at leastone inverted abasic moiety; A is conjugated anywhere on B, C, or D; B isconjugated anywhere on A, C, or D; C is conjugated anywhere on A, B, orD; and D is conjugated anywhere on A, B, or C.

In some embodiments, a molecule (e.g., a therapeutic molecule) describedherein comprises a molecule according to Formula (Ma): A-X—B-L-D-Y—C.

In some embodiments, a molecule (e.g., a therapeutic molecule) describedherein comprises a molecule according to Formula (IIIb):A_(a)-X—B_(b)-L-D_(n).

In some embodiments, a molecule (e.g., a therapeutic molecule) describedherein comprises a molecule according to Formula (IV):A-X—(B_(b)—Y—C_(c)-L-D_(n))_(m)wherein,

A is a binding moiety;

B is a polynucleotide;

C is a polymer;

D is an endosomolytic moiety;

X is a bond or first linker;

Y is a bond or second linker;

L is a bond or third linker;

a and b are independently an integer between 1-3;

c is an integer between 0 and 3;

n is an integer between 0 and 10; and

m is an integer between 1-3; and

wherein the polynucleotide comprises at least one 2′ modifiednucleotide, at least one modified internucleotide linkage, or at leastone inverted abasic moiety; C is conjugated anywhere on B or D; and D isconjugated anywhere on B or C.

In some embodiments, a molecule (e.g., a therapeutic molecule) describedherein comprises a molecule according to Formula (IVa):A-X—(B_(b)-L-D_(n)-Y—C_(c))_(m).

In some embodiment, a molecule (e.g., a therapeutic molecule) describedherein is a molecule as illustrated in FIGS. 1A-1T. In some instances, amolecule (e.g., a therapeutic molecule) described herein is a moleculeas illustrated in FIGS. 1A-1G. In some cases, a molecule (e.g., atherapeutic molecule) described herein is a molecule as illustrated inFIGS. 1H-1N. In additional cases, a molecule (e.g., a therapeuticmolecule) described herein is a molecule as illustrated in FIGS. 1O-1T.

In some embodiments, a molecule (e.g., a therapeutic molecule) is amolecule as illustrated in FIG. 1A.

In some embodiments, a molecule (e.g., a therapeutic molecule) is amolecule as illustrated in FIG. 1B.

In some embodiments, a molecule (e.g., a therapeutic molecule) is amolecule as illustrated in FIG. 1C.

In some embodiments, a molecule (e.g., a therapeutic molecule) is amolecule as illustrated in FIG. 1D.

In some embodiments, a molecule (e.g., a therapeutic molecule) is amolecule as illustrated in FIG. 1E.

In some embodiments, a molecule (e.g., a therapeutic molecule) is amolecule as illustrated in FIG. 1F.

In some embodiments, a molecule (e.g., a therapeutic molecule) is amolecule as illustrated in FIG. 1G.

In some embodiments, a molecule (e.g., a therapeutic molecule) is amolecule as illustrated in FIG. 1H.

In some embodiments, a molecule (e.g., a therapeutic molecule) is amolecule as illustrated in FIG. 1I.

In some embodiments, a molecule (e.g., a therapeutic molecule) is amolecule as illustrated in FIG. 1H.

In some embodiments, a molecule (e.g., a therapeutic molecule) is amolecule as illustrated in FIG. 1K.

In some embodiments, a molecule (e.g., a therapeutic molecule) is amolecule as illustrated in FIG. 1L.

In some embodiments, a molecule (e.g., a therapeutic molecule) is amolecule as illustrated in FIG. 1M.

In some embodiments, a molecule (e.g., a therapeutic molecule) is amolecule as illustrated in FIG. 1N.

In some embodiments, a molecule (e.g., a therapeutic molecule) is amolecule as illustrated in FIG. 1O.

In some embodiments, a molecule (e.g., a therapeutic molecule) is amolecule as illustrated in FIG. 1P.

In some embodiments, a molecule (e.g., a therapeutic molecule) is amolecule as illustrated in FIG. 1Q.

In some embodiments, a molecule (e.g., a therapeutic molecule) is amolecule as illustrated in FIG. 1R.

The antibody as illustrated in FIGS. 1A-1R is for representationpurposes only and encompasses a humanized antibody or binding fragmentthereof, chimeric antibody or binding fragment thereof, monoclonalantibody or binding fragment thereof, monovalent Fab′, divalent Fab2,single-chain variable fragment (scFv), diabody, minibody, nanobody,single-domain antibody (sdAb), or camelid antibody or binding fragmentthereof.

Polynucleic Acid Molecule Targets

In some embodiments, the polynucleic acid molecule B is a polynucleicacid molecule (or polynucleotide) that hybridizes to a target region onan oncogene. In some instances, oncogenes are further classified intoseveral categories: growth factors or mitogens, receptor tyrosinekinases, cytoplasmic tyrosine kinases, cytoplasmic serine/threoninekinases, regulatory GTPases, and transcription factors. Exemplary growthfactors include c-Sis. Exemplary receptor tyrosine kinases includeepidermal growth factor receptor (EGFR), platelet-derived growth factorreceptor (PDGFR), vascular endothelial growth factor receptor (VEGFR),and HER2/neu. Exemplary cytoplasmic tyrosine kinases include Src-familytyrosine kinases, Syk-ZAP-70 family of tyrosine kinases, BTK family oftyrosine kinases, and Abl gene in CML. Exemplary cytoplasmicserine/threonine kinases include Raf kinase and cyclin-dependentkinases. Exemplary regulatory GTPases include Ras family of proteinssuch as KRAS. Exemplary transcription factors include MYC gene. In someinstances, an oncogene described herein comprises an oncogene selectedfrom growth factors or mitogens, receptor tyrosine kinases, cytoplasmictyrosine kinases, cytoplasmic serine/threonine kinases, regulatoryGTPases, or transcription factors. In some embodiments, the polynucleicacid molecule is a polynucleic acid molecule that hybridizes to a targetregion of an oncogene selected from growth factors or mitogens, receptortyrosine kinases, cytoplasmic tyrosine kinases, cytoplasmicserine/threonine kinases, regulatory GTPases, or transcription factors.

In some embodiments, an oncogene described herein comprises Abl, AKT-2,ALK, AML1 (or RUNX1), AR, AXL, BCL-2, 3, 6, BRAF, c-MYC, EGFR, ErbB-2(Her2, Neu), Fms, FOS, GLI1, HPRT1, IL-3, INTS2, JUN, KIT, KS3, K-sam,LBC (AKAP13), LCK, LMO1, LMO2, LYL1, MAS1, MDM2, MET, MLL (KMT2A), MOS,MYB, MYH11/CBFB, NOTCH1 (TAN1), NTRK1 (TRK), OST (SLC51B), PAX5, PIM1,PRAD-1, RAF, RAR/PML, HRAS, KRAS, NRAS, REL/NRG, RET, ROS, SKI, SRC,TIAM1, or TSC2. In some embodiments, the polynucleic acid molecule is apolynucleic acid molecule that hybridizes to a target region of Abl,AKT-2, ALK, AML1 (or RUNX1), AR, AXL, BCL-2, 3, 6, BRAF, c-MYC, EGFR,ErbB-2 (Her2, Neu), Fms, FOS, GLI1, HPRT1, IL-3, INTS2, JUN, KIT, KS3,K-sam, LBC (AKAP13), LCK, LMO1, LMO2, LYL1, MAS1, MDM2, MET, MLL(KMT2A), MOS, MYB, MYH11/CBFB, NOTCH1 (TAN1), NTRK1 (TRK), OST (SLC51B),PAX5, PIM1, PRAD-1, RAF, RAR/PML, HRAS, KRAS, NRAS, REL/NRG, RET, ROS,SKI, SRC, TIAM1, or TSC2.

In some embodiments, an oncogene described herein comprises KRAS, EGFR,AR, HPRT1, CNNTB1 (β-catenin), or β-catenin associated genes. In someembodiments, the polynucleic acid molecule B is a polynucleic acidmolecule that hybridizes to a target region of KRAS, EGFR, AR, HPRT1,CNNTB1 (β-catenin), or β-catenin associated genes. In some embodiments,the polynucleic acid molecule B is a polynucleic acid molecule thathybridizes to a target region of KRAS. In some embodiments, thepolynucleic acid molecule B is a polynucleic acid molecule thathybridizes to a target region of EGFR. In some embodiments, thepolynucleic acid molecule B is a polynucleic acid molecule thathybridizes to a target region of AR. In some embodiments, thepolynucleic acid molecule B is a polynucleic acid molecule thathybridizes to a target region of CNNTB1 (β-catenin). In someembodiments, the polynucleic acid molecule B is a polynucleic acidmolecule that hybridizes to a target region of CNNTB1 (β-catenin)associated genes. In some instances, the β-catenin associated genescomprise PIK3CA, PIK3CB, and Myc. In some instances, the polynucleicacid molecule B is a polynucleic acid molecule that hybridizes to atarget region of HPRT1.

Polynucleic Acid Molecules that Target Kirsten Rat Sarcoma ViralOncogene Homolog (KRAS)

Kirsten Rat Sarcoma Viral Oncogene Homolog (also known as GTPase KRas,V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog, or KRAS) isinvolved in regulating cell division. The K-Ras protein is a GTPasebelonging to the Ras superfamily. In some instances, K-Ras modulatescell cycle progression, as well as induces growth arrest, apoptosis, andreplicative senescence under different environmental triggers (e.g.,cellular stress, ultraviolet, heat shock, or ionizing irradiation). Insome cases, wild type KRAS gene has been shown to be frequently lostduring tumor progression in different types of cancer, while mutationsof KRAS gene have been linked to cancer development. In some instances,KRAS amplification has also been implicated in cancer development (see,for example, Valtorta et al. “KRAS gene amplification in colorectalcancer and impact on response to EGFR-targeted therapy,” Int. J. Cancer133: 1259-1266 (2013)). In such cases, the cancer pertains to arefractory cancer in which the patient has acquired resistance to aparticular inhibitor or class of inhibitors.

In some embodiments, the KRAS gene is wild type or comprises a mutation.In some instances, KRAS mRNA is wild type or comprises a mutation. Insome instances, the polynucleic acid molecule is a polynucleic acidmolecule that hybridizes to a target region of wild type KRAS DNA orRNA. In some instances, the polynucleic acid molecule is a polynucleicacid molecule that hybridizes to a target region of KRAS DNA or RNAcomprising a mutation (e.g., a substitution, a deletion, or anaddition).

In some embodiments, KRAS DNA or RNA comprises one or more mutations. Insome embodiments, KRAS DNA or RNA comprises one or more mutations atcodons 12 or 13 in exon 1. In some instances, KRAS DNA or RNA comprisesone or more mutations at codons 61, 63, 117, 119, or 146. In someinstances, KRAS DNA or RNA comprises one or more mutations at positionscorresponding to amino acid residues 12, 13, 18, 19, 20, 22, 24, 26, 36,59, 61, 63, 64, 68, 110, 116, 117, 119, 146, 147, 158, 164, 176, or acombination thereof of the KRAS polypeptide. In some embodiments, KRASDNA or RNA comprises one or more mutations at positions corresponding toamino acid residues selected from G12V, G12D, G12C, G12A, G12S, G12F,G13C, G13D, G13V, A18D, L19F, T20R, Q22K, I24N, N26K, I36L, I36M, A59G,A59E, Q61K, Q61H, Q61L, Q61R, E63K, Y64D, Y64N, R68S, P110S, K117N,C118S, A146T, A146P, A146V, K147N, T158A, R164Q, K176Q, or a combinationthereof of the KRAS polypeptide.

In some embodiments, the polynucleic acid molecule hybridizes to atarget region of KRAS DNA or RNA comprising one or more mutations. Insome embodiments, the polynucleic acid molecule hybridizes to a targetregion of KRAS DNA or RNA comprising one or more mutations at codons 12or 13 in exon 1. In some embodiments, the polynucleic acid moleculehybridizes to a target region of KRAS DNA or RNA comprising one or moremutations at codons 61, 63, 117, 119, or 146. In some embodiments, thepolynucleic acid molecule hybridizes to a target region of KRAS DNA orRNA comprising one or more mutations at positions corresponding to aminoacid residues 12, 13, 18, 19, 20, 22, 24, 26, 36, 59, 61, 63, 64, 68,110, 116, 117, 119, 146, 147, 158, 164, 176, or a combination thereof ofthe KRAS polypeptide. In some embodiments, the polynucleic acid moleculehybridizes to a target region of KRAS DNA or RNA comprising one or moremutations corresponding to amino acid residues selected from G12V, G12D,G12C, G12A, G12S, G12F, G13C, G13D, G13V, A18D, L19F, T20R, Q22K, I24N,N26K, I36L, I36M, A59G, A59E, Q61K, Q61H, Q61L, Q61R, E63K, Y64D, Y64N,R68S, P110S, K117N, C118S, A146T, A146P, A146V, K147N, T158A, R164Q,K176Q, or a combination thereof of the KRAS polypeptide.

Polynucleic Acid Molecules that Target Epidermal Growth Factor Receptor(EGFR)

Epidermal growth factor receptor (EGFR, ErbB-1, or HER1) is atransmembrane tyrosine kinase receptor and a member of the ErbB familyof receptors, which also include HER2/c-neu (ErbB-2), Her3 (ErbB-3) andHer4 (ErbB-4). In some instances, EGFR mutations drive the downstreamactivation of RAS/RAF/MAPK, PI3K/AKT, and/or JAK/STAT pathways, leadingto mitosis, cell proliferation, and suppression of apoptosis. Inaddition, amplification of wild-type EGFR gene has been implicated inthe development of cancers such as glioblastomas and non-small cell lungcancer (Talasila, et al., “EGFR Wild-type Amplification and ActivationPromote Invasion and Development of Glioblastoma Independent ofAngiogenesis,” Acta Neuropathol. 125(5): 683-698 (2013); Bell et al.,“Epidermal Growth Factor Receptor Mutations and Gene Amplification inNon-Small-Cell Lung Cancer: Molecular Analysis of the IDEAL/INTACTGefitinib Trials,” J. Clinical Oncology 23(31): 8081-8092 (2005)).

In some embodiments, EGFR DNA or RNA is wild type EGFR or EGFRcomprising a mutation. In some instances, EGFR is wild type EGFR. Insome instances, EGFR DNA or RNA comprises a mutation. In some instances,the polynucleic acid molecule hybridizes to a target region of wild typeEGFR DNA or RNA. In some instances, the polynucleic acid moleculehybridizes to a target region of EGFR DNA or RNA comprising a mutation(e.g., a substitution, a deletion, or an addition).

In some instances, EGFR DNA or RNA comprises one or more mutations. Insome embodiments, EGFR DNA or RNA comprises one or more mutations withinone or more exons. In some instances, the one or more exons compriseexon 18, exon 19, exon 20, exon 21 or exon 22. In some instances, EGFRDNA or RNA comprises one or more mutations in exon 18, exon 19, exon 20,exon 21, exon 22 or a combination thereof.

In some instances, EGFR DNA or RNA comprises one or more mutations atpositions corresponding to amino acid residues 34, 38, 45, 62, 63, 77,78, 108, 114, 120, 140, 148, 149, 160, 177, 178, 189, 191, 198, 220,222, 223, 229, 237, 240, 244, 252, 254, 255, 256, 263, 270, 273, 276,282, 288, 289, 301, 303, 304, 309, 314, 326, 331, 354, 363, 373, 337,380, 384, 393, 427, 428, 437, 441, 447, 465, 475, 515, 526, 527, 531,536, 541, 546, 571, 588, 589, 596, 596, 598, 602, 614, 620, 628, 636,641, 645, 651, 671, 689, 694, 700, 709, 712, 714, 715, 716, 719, 720,721, 731, 733, 739-744, 742, 746-750, 746-752, 746, 747, 747-749,747-751, 747-753, 751, 752, 754, 752-759, 750, 761-762, 761, 763, 765,767-768, 767-769, 768, 769, 769-770, 770-771, 772, 773-774, 773, 774,774-775, 776, 779, 783, 784, 786, 790, 792, 794, 798, 803, 805, 807,810, 826, 827, 831, 832, 833, 835, 837, 838, 839, 842, 843, 847, 850,851, 853, 854, 856, 858, 861, 863, 894, 917, 967, 1006, 1019, 1042,1100, 1129, 1141, 1153, 1164, 1167, or a combination thereof of the EGFRpolypeptide. In some embodiments, EGFR DNA or RNA comprises one or moremutations at positions corresponding to amino acid residues 747, 761,790, 854, 858, or a combination thereof of the EGFR polypeptide. In someembodiments, EGFR DNA or RNA comprises one or more mutations atpositions corresponding to amino acid residues 761, 790, 858, or acombination thereof of the EGFR polypeptide. In some embodiments, EGFRDNA or RNA comprises a mutation at a position corresponding to aminoacid residue 747 of the EGFR polypeptide. In some embodiments, EGFR DNAor RNA comprises a mutation at a position corresponding to amino acidresidue 761 of the EGFR polypeptide. In some embodiments, EGFR DNA orRNA comprises a mutation at a position corresponding to amino acidresidue 790 of the EGFR polypeptide. In some embodiments, EGFR DNA orRNA comprises a mutation at a position corresponding to amino acidresidue 854 of the EGFR polypeptide. In some embodiments, EGFR DNA orRNA comprises a mutation at a position corresponding to amino acidresidue 858 of the EGFR polypeptide.

In some embodiments, EGFR DNA or RNA comprises one or more mutationsselected from T34M, L38V, E45Q, L62R, G63R, G63K, S77F, F78L, R108K,R108G, E114K, A120P, L140V, V148M, R149W, E160K, S177P, M178I, K189T,D191N, S198R, S220P, R222L, R222C, S223Y, S229C, A237Y, C240Y, R244G,R252C, R252P, F254I, 8255 (nonsense mutation), D256Y, T263P, Y270C,T273A, Q276 (nonsense), E282K, G288 (frame shift), A289D, A289V, A289T,A289N, A289D, V301 (deletion), D303H, H304Y, R309Q, D314N, C326R, G331R,T354M, T363I, P373Q, R337S, 5380 (frame shift), T384S, D393Y, R427L,G428S, S437Y, V441I, S447Y, G465R, I475V, C515S, C526S, R527L, R531(nonsense), V536M, L541I, P546Q, C571S, G588S, P589L, P596L, P596S,P596R, P596L, G598V, G598A, E602G, G614D, C620Y, C620W, C628Y, C628F,C636Y, T638M, P641H, S645C, V651M, R671C, V689M, P694S, N700D, E709A,E709K, E709Q, E709K, F712L, K714N, I715S, K716R, G719A, G719C, G719D,G719S, S720C, S720F, G721V, W731Stop, P733L, K739-1744 (insertion),V742I, V742A, E746-A750 (deletion), E746K, L747S, L747-E749 (deletion),L747-T751 (deletion), L747-P753 (deletion), G746-S752 (deletion), T751I,S752Y, K754 (deletion), S752-1759 (deletion), A750P, D761-E762 (e.g.,residues EAFQ insertion (SEQ ID NO: 2110)), D761N, D761Y, A763V, V765A,A767-S768 (e.g., residues TLA insertion), A767-V769 (e.g., residues ASVinsertion), S768I, S768T, V769L, V769M, V769-D770 (e.g., residue Yinsertion), 770-771 (e.g., residues GL insertion), 770-771 (e.g.,residue G insertion), 770-771 (e.g., residues CV insertion), 770-771(e.g., residues SVD insertion), P772R, 773-774 (e.g., residues NPHinsertion), H773R, H773L, V774M, 774-775 (e.g., residues HV insertion),R776H, R776C, G779F, T783A, T784F, T854A, V786L, T790M, L792P, P794H,L798F, R803W, H805R, D807H, G810S, N826S, Y827 (nonsense), R831H, R832C,R832H, L833F, L833V, H835L, D837V, L838M, L838P, A839V, N842H, V843L,T847K, T847I, H850N, V851A, I853T, F856L, L858R, L858M, L861Q, L861R,G863D, Q894L, G917A, E967A, D1006Y, P1019L, S1042N, R1100S, H1129Y,T1141S, S1153I, Q1164R, L1167M, or a combination thereof of the EGFRpolypeptide.

In some instances, the polynucleic acid molecule hybridizes to a targetregion of EGFR DNA or RNA comprising one or more mutations. In someembodiments, the polynucleic acid molecule hybridizes to a target regionof EGFR DNA or RNA comprising one or more mutations in exon 18, exon 19,exon 20, exon 21, exon 22 or a combination thereof.

In some embodiments, the polynucleic acid molecule hybridizes to atarget region of EGFR DNA or RNA comprising one or more mutations atpositions corresponding to amino acid residues 34, 38, 45, 62, 63, 77,78, 108, 114, 120, 140, 148, 149, 160, 177, 178, 189, 191, 198, 220,222, 223, 229, 237, 240, 244, 252, 254, 255, 256, 263, 270, 273, 276,282, 288, 289, 301, 303, 304, 309, 314, 326, 331, 354, 363, 373, 337,380, 384, 393, 427, 428, 437, 441, 447, 465, 475, 515, 526, 527, 531,536, 541, 546, 571, 588, 589, 596, 596, 598, 602, 614, 620, 628, 636,641, 645, 651, 671, 689, 694, 700, 709, 712, 714, 715, 716, 719, 720,721, 731, 733, 739-744, 742, 746-750, 746-752, 746, 747, 747-749,747-751, 747-753, 751, 752, 754, 752-759, 750, 761-762, 761, 763, 765,767-768, 767-769, 768, 769, 769-770, 770-771, 772, 773-774, 773, 774,774-775, 776, 779, 783, 784, 786, 790, 792, 794, 798, 803, 805, 807,810, 826, 827, 831, 832, 833, 835, 837, 838, 839, 842, 843, 847, 850,851, 853, 854, 856, 858, 861, 863, 894, 917, 967, 1006, 1019, 1042,1100, 1129, 1141, 1153, 1164, 1167, or a combination thereof of the EGFRpolypeptide. In some embodiments, the polynucleic acid moleculehybridizes to a target region of EGFR DNA or RNA comprising one or moremutations at positions corresponding to amino acid residues 747, 761,790, 854, 858, or a combination thereof of the EGFR polypeptide. In someembodiments, the polynucleic acid molecule hybridizes to a target regionof EGFR DNA or RNA comprising one or more mutations at positionscorresponding to amino acid residues 761, 790, 858, or a combinationthereof of the EGFR polypeptide. In some embodiments, the polynucleicacid molecule hybridizes to a target region of EGFR DNA or RNAcomprising a mutation at a position corresponding to amino acid residue747 of the EGFR polypeptide. In some embodiments, the polynucleic acidmolecule hybridizes to a target region of EGFR DNA or RNA comprising amutation at a position corresponding to amino acid residue 761 of theEGFR polypeptide. In some embodiments, the polynucleic acid moleculehybridizes to a target region of EGFR DNA or RNA comprising a mutationat a position corresponding to amino acid residue 790 of the EGFRpolypeptide. In some embodiments, the polynucleic acid moleculehybridizes to a target region of EGFR DNA or RNA comprising a mutationat a position corresponding to amino acid residue 854 of the EGFRpolypeptide. In some embodiments, the polynucleic acid moleculehybridizes to a target region of EGFR DNA or RNA comprising a mutationat a position corresponding to amino acid residue 858 of the EGFRpolypeptide.

In some embodiments, the polynucleic acid molecule hybridizes to atarget region of EGFR DNA or RNA comprising one or more mutationsselected from T34M, L38V, E45Q, L62R, G63R, G63K, S77F, F78L, R108K,R108G, E114K, A120P, L140V, V148M, R149W, E160K, S177P, M178I, K189T,D191N, S198R, S220P, R222L, R222C, S223Y, S229C, A237Y, C240Y, R244G,R252C, R252P, F254I, R255 (nonsense mutation), D256Y, T263P, Y270C,T273A, Q276 (nonsense), E282K, G288 (frame shift), A289D, A289V, A289T,A289N, A289D, V301 (deletion), D303H, H304Y, R309Q, D314N, C326R, G331R,T354M, T363I, P373Q, R337S, S380 (frame shift), T384S, D393Y, R427L,G428S, S437Y, V441I, S447Y, G465R, I475V, C515S, C526S, R527L, R531(nonsense), V536M, L541I, P546Q, C571S, G588S, P589L, P596L, P596S,P596R, P596L, G598V, G598A, E602G, G614D, C620Y, C620W, C628Y, C628F,C636Y, T638M, P641H, S645C, V651M, R671C, V689M, P694S, N700D, E709A,E709K, E709Q, E709K, F712L, K714N, I715S, K716R, G719A, G719C, G719D,G719S, S720C, S720F, G721V, W731Stop, P733L, K739-1744 (insertion),V742I, V742A, E746-A750 (deletion), E746K, L747S, L747-E749 (deletion),L747-T751 (deletion), L747-P753 (deletion), G746-S752 (deletion), T751I,S752Y, K754 (deletion), S752-1759 (deletion), A750P, D761-E762 (e.g.,residues EAFQ insertion (SEQ ID NO: 2110)), D761N, D761Y, A763V, V765A,A767-5768 (e.g., residues TLA insertion), A767-V769 (e.g., residues ASVinsertion), S768I, S768T, V769L, V769M, V769-D770 (e.g., residue Yinsertion), 770-771 (e.g., residues GL insertion), 770-771 (e.g.,residue G insertion), 770-771 (e.g., residues CV insertion), 770-771(e.g., residues SVD insertion), P772R, 773-774 (e.g., residues NPHinsertion), H773R, H773L, V774M, 774-775 (e.g., residues HV insertion),R776H, R776C, G779F, T783A, T784F, T854A, V786L, T790M, L792P, P794H,L798F, R803W, H805R, D807H, G810S, N826S, Y827 (nonsense), R831H, R832C,R832H, L833F, L833V, H835L, D837V, L838M, L838P, A839V, N842H, V843L,T847K, T847I, H850N, V851A, I853T, F856L, L858R, L858M, L861Q, L861R,G863D, Q894L, G917A, E967A, D1006Y, P1019L, S1042N, R1100S, H1129Y,T1141S, S1153I, Q1164R, L1167M, or a combination thereof of the EGFRpolypeptide. In some embodiments, the polynucleic acid moleculehybridizes to a target region of EGFR DNA or RNA comprising one or moremutations selected from L747S, D761Y, T790M, T854A, L858R, or acombination thereof of the EGFR polypeptide. In some embodiments, thepolynucleic acid molecule hybridizes to a target region of EGFR DNA orRNA comprising one or more mutations selected from D761Y, T790M, L858R,or a combination thereof of the EGFR polypeptide. In some embodiments,the polynucleic acid molecule hybridizes to a target region of EGFR DNAor RNA comprising mutation L747S of the EGFR polypeptide. In someembodiments, the polynucleic acid molecule hybridizes to a target regionof EGFR DNA or RNA comprising mutation D761Y of the EGFR polypeptide. Insome embodiments, the polynucleic acid molecule hybridizes to a targetregion of EGFR DNA or RNA comprising mutation T790M of the EGFRpolypeptide. In some embodiments, the polynucleic acid moleculehybridizes to a target region of EGFR DNA or RNA comprising mutationT854A of the EGFR polypeptide. In some embodiments, the polynucleic acidmolecule hybridizes to a target region of EGFR DNA or RNA comprisingmutation L858R of the EGFR polypeptide.

Polynucleic Acid Molecules that Target Androgen Receptor (AR)

Androgen receptor (AR) (also known as NR3C4, nuclear receptor subfamily3, group C, gene 4) belongs to the steroid hormone group of nuclearreceptor superfamily along with related members: estrogen receptor (ER),glucocorticoid receptor (GR), progesterone receptor (PR), andmineralocorticoid receptor (MR). Androgens, or steroid hormones,modulate protein synthesis and tissue remodeling through the androgenreceptor. The AR protein is a ligand-inducible zinc finger transcriptionfactor that regulates target gene expression. The presence of mutationsin the AR gene has been observed in several types of cancers (e.g.,prostate cancer, breast cancer, bladder cancer, or esophageal cancer),and in some instances, has been linked to metastatic progression.

In some embodiments, AR DNA or RNA is wild type or comprises one or moremutations and/or splice variants. In some instances, AR DNA or RNAcomprises one or more mutations. In some instances, AR DNA or RNAcomprises one or more splice variants selected from AR splice variantsincluding but not limited to AR1/2/2b, ARV2, ARV3, ARV4, AR1/2/3/2b,ARV5, ARV6, ARV7, ARV9, ARV10, ARV11, ARV12, ARV13, ARV14, ARV15, ARV16,and ARV(v567es). In some instances, the polynucleic acid moleculehybridizes to a target region of AR DNA or RNA comprising a mutation(e.g., a substitution, a deletion, or an addition) or a splice variant.

In some embodiments, AR DNA or RNA comprises one or more mutations. Insome embodiments, AR DNA or RNA comprises one or more mutations withinone or more exons. In some instances, the one or more exons compriseexon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, or exon 8. Insome embodiments, AR DNA or RNA comprises one or more mutations withinexon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8 or acombination thereof. In some instances, AR DNA or RNA comprises one ormore mutations at positions corresponding to amino acid residues 2, 14,16, 29, 45, 54, 57, 64, 106, 112, 176, 180, 184, 194, 198, 204, 214,221, 222, 233, 243, 252, 255, 266, 269, 287, 288, 334, 335, 340, 363,368, 369, 390, 403, 443, 491, 505, 513, 524, 524, 528, 533, 547, 548,564, 567, 568, 574, 547, 559, 568, 571, 573, 575, 576, 577, 578, 579,580, 581, 582, 585, 586, 587, 596, 597, 599, 601, 604, 607, 608, 609,610, 611, 615, 616, 617, 619, 622, 629, 630, 638, 645, 647, 653, 662,664, 670, 671, 672, 674, 677, 681, 682, 683, 684, 687, 688, 689, 690,695, 700, 701, 702, 703, 705, 706, 707, 708, 710, 711, 712, 715, 717,720, 721, 722, 723, 724, 725, 726, 727, 728, 730, 732, 733, 737, 739,741, 742, 743, 744, 745, 746, 748, 749, 750, 751, 752, 754, 755, 756,757, 758, 759, 762, 763, 764, 765, 766, 767, 768, 771, 772, 774, 777,779, 786, 795, 780, 782, 784, 787, 788, 790, 791, 793, 794, 798, 802,803, 804, 806, 807, 812, 813, 814, 819, 820, 821, 824, 827, 828, 830,831, 834, 840, 841, 842, 846, 854, 855, 856, 863, 864, 866, 869, 870,871, 874, 875, 877, 879, 880, 881, 886, 888, 889, 891, 892, 895, 896,897, 898, 902, 903, 904, 907, 909, 910, 911, 913, 916, 919, or acombination thereof of the AR polypeptide. In some embodiments, AR DNAor RNA comprises one or more mutations at positions corresponding toamino acid residues selected from E2K, P14Q, K16N, V29M, S45T, L54S,L57Q, Q64R, Y106C, Q112H, S176S, K180R, L184P, Q194R, E198G, G204S,G214R, K221N, N222D, D233K, S243L, A252V, L255P, M266T, P269S, A287D,E288K, S334P, S335T, P340L, Y363N, L368V, A369P, P390R, P390S, P390L,A403V, Q443R, G491S, G505D, P513S, G524D, G524S, D528G, P533S, L547F,P548S, D564Y, S567F, G568W, L574P, L547F, C559Y, G568W, G568V, Y571C,Y571H, A573D, T575A, C576R, C576F, G577R, S578T, C579Y, C579F, K580R,V581F, F582Y, F582S, R585K, A586V, A587S, A596T, A596S, S597G, S597I,N599Y, C601F, D604Y, R607Q, R608K, K609N, D610T, C611Y, R615H, R615P,R615G, R616C, L616R, L616P, R617P, C619Y, A622V, R629W, R629Q, K630T,L638M, A645D, S647N, E653K, S662 (nonsense), I664N, Q670L, Q670R, P671H,I672T, L674P, L677P, E681L, P682T, G683A, V684I, V684A, A687V, G688Q,H689P, D690V, D695N, D695V, D695H, L700M, L701P, L701I, H701H, S702A,S703G, N705S, N705Y, E706 (nonsense), L707R, G708A, R710T, Q711E, L712F,V715M, K717Q, K720E, A721T, L722F, P723S, G724S, G724D, G724N, F725L,R726L, N727K, L728S, L728I, V730M, D732N, D732Y, D732E, Q733H, I737T,Y739D, W741R, M742V, M742I, G743R, G743V, L744F, M745T, V746M, A748D,A748V, A748T, M749V, M749I, G750S, G750D, W751R, R752Q, F754V, F754L,T755A, N756S, N756D, V757A, N758T, S759F, S759P, L762F, Y763H, Y763C,F764L, A765T, A765V, P766A, P766S, D767E, L768P, L768M, N771H, E772G,E772A, R774H, R774C, K777T, R779W, R786Q, G795V, M780I, S782N, C784Y,M787V, R788S, L790F, S791P, E793D, F794S, Q798E, Q802R, G803L, F804L,C806Y, M807V, M807R, M807I, L812P, F813V, S814N, N819Q, G820A, L821V,Q824L, Q824R, F827L, F827V, D828H, L830V, L830P, R831Q, R831L, Y834C,R840C, R840H, I841S, I842T, R846G, R854K, R855C, R855H, F856L, L863R,D864N, D864E, D864G, V866L, V866M, V866E, I869M, A870G, A870V, R871G,H874Y, H874R, Q875K, T877S, T877A, D879T, D879G, L880Q, L881V, M886V,S888L, V889M, F891L, P892L, M895T, A896T, E897D, I898T, Q902R, V903M,P904S, P904H, L907F, G909R, G909E, K910R, V911L, P913S, F916L, Q919R, ora combination thereof of the AR polypeptide.

In some embodiments, the polynucleic acid molecule hybridizes to atarget region of AR DNA or RNA comprising one or more mutations. In someembodiments the polynucleic acid hybridizes to one or more AR splicevariants. In some embodiments the polynucleic acid hybridizes to AR DNAor RNA comprising one or more AR splice variants including but notlimited to AR1/2/2b, ARV2, ARV3, ARV4, AR1/2/3/2b, ARV5, ARV6, ARV7,ARV9, ARV10, ARV11, ARV12, ARV13, ARV14, ARV15, ARV16, and ARV(v567es).In some embodiments, the polynucleic acid molecule hybridizes to atarget region of AR DNA or RNA comprising one or more mutations withinexon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8 or acombination thereof. In some embodiments, the polynucleic acid moleculehybridizes to a target region of AR DNA or RNA comprising one or moremutations at positions corresponding to amino acid residues 2, 14, 16,29, 45, 54, 57, 64, 106, 112, 176, 180, 184, 194, 198, 204, 214, 221,222, 233, 243, 252, 255, 266, 269, 287, 288, 334, 335, 340, 363, 368,369, 390, 403, 443, 491, 505, 513, 524, 524, 528, 533, 547, 548, 564,567, 568, 574, 547, 559, 568, 571, 573, 575, 576, 577, 578, 579, 580,581, 582, 585, 586, 587, 596, 597, 599, 601, 604, 607, 608, 609, 610,611, 615, 616, 617, 619, 622, 629, 630, 638, 645, 647, 653, 662, 664,670, 671, 672, 674, 677, 681, 682, 683, 684, 687, 688, 689, 690, 695,700, 701, 702, 703, 705, 706, 707, 708, 710, 711, 712, 715, 717, 720,721, 722, 723, 724, 725, 726, 727, 728, 730, 732, 733, 737, 739, 741,742, 743, 744, 745, 746, 748, 749, 750, 751, 752, 754, 755, 756, 757,758, 759, 762, 763, 764, 765, 766, 767, 768, 771, 772, 774, 777, 779,786, 795, 780, 782, 784, 787, 788, 790, 791, 793, 794, 798, 802, 803,804, 806, 807, 812, 813, 814, 819, 820, 821, 824, 827, 828, 830, 831,834, 840, 841, 842, 846, 854, 855, 856, 863, 864, 866, 869, 870, 871,874, 875, 877, 879, 880, 881, 886, 888, 889, 891, 892, 895, 896, 897,898, 902, 903, 904, 907, 909, 910, 911, 913, 916, 919, or a combinationthereof of the AR polypeptide. In some embodiments, the polynucleic acidmolecule hybridizes to a target region of AR DNA or RNA comprising oneor more mutations selected from E2K, P14Q, K16N, V29M, S45T, L54S, L57Q,Q64R, Y106C, Q112H, S176S, K180R, L184P, Q194R, E198G, G204S, G214R,K221N, N222D, D233K, S243L, A252V, L255P, M266T, P269S, A287D, E288K,S334P, S335T, P340L, Y363N, L368V, A369P, P390R, P390S, P390L, A403V,Q443R, G491S, G505D, P513S, G524D, G524S, D528G, P533S, L547F, P548S,D564Y, S567F, G568W, L574P, L547F, C559Y, G568W, G568V, Y571C, Y571H,A573D, T575A, C576R, C576F, G577R, S578T, C579Y, C579F, K580R, V581F,F582Y, F582S, R585K, A586V, A587S, A596T, A596S, S597G, S597I, N599Y,C601F, D604Y, R607Q, R608K, K609N, D610T, C611Y, R615H, R615P, R615G,R616C, L616R, L616P, R617P, C619Y, A622V, R629W, R629Q, K630T, L638M,A645D, S647N, E653K, S662 (nonsense), I664N, Q670L, Q670R, P671H, I672T,L674P, L677P, E681L, P682T, G683A, V684I, V684A, A687V, G688Q, H689P,D690V, D695N, D695V, D695H, L700M, L701P, L701I, H701H, S702A, S703G,N705S, N705Y, E706 (nonsense), L707R, G708A, R710T, Q711E, L712F, V715M,K717Q, K720E, A721T, L722F, P723S, G724S, G724D, G724N, F725L, R726L,N727K, L728S, L728I, V730M, D732N, D732Y, D732E, Q733H, I737T, Y739D,W741R, M742V, M742I, G743R, G743V, L744F, M745T, V746M, A748D, A748V,A748T, M749V, M749I, G750S, G750D, W751R, R752Q, F754V, F754L, T755A,N756S, N756D, V757A, N758T, S759F, S759P, L762F, Y763H, Y763C, F764L,A765T, A765V, P766A, P766S, D767E, L768P, L768M, N771H, E772G, E772A,R774H, R774C, K777T, R779W, R786Q, G795V, M780I, S782N, C784Y, M787V,R788S, L790F, S791P, E793D, F794S, Q798E, Q802R, G803L, F804L, C806Y,M807V, M807R, M807I, L812P, F813V, S814N, N819Q, G820A, L821V, Q824L,Q824R, F827L, F827V, D828H, L830V, L830P, R831Q, R831L, Y834C, R840C,R840H, I841S, I842T, R846G, R854K, R855C, R855H, F856L, L863R, D864N,D864E, D864G, V866L, V866M, V866E, I869M, A870G, A870V, R871G, H874Y,H874R, Q875K, T877S, T877A, D879T, D879G, L880Q, L881V, M886V, S888L,V889M, F891L, P892L, M895T, A896T, E897D, I898T, Q902R, V903M, P904S,P904H, L907F, G909R, G909E, K910R, V911L, P913S, F916L, Q919R, or acombination thereof of the AR polypeptide.

Polynucleic Acid Molecules that Target B-Catenin andB-Catenin-Associated Genes

Catenin beta-1 (also known as CTNNB1, β-catenin, or beta-catenin) is amember of the catenin protein family. In humans, it is encoded by theCTNNB1 gene and is known for its dual functions—cell-cell adhesion andgene transcription. Beta-catenin is an integral structural component ofcadherin-based adherens junctions and regulates cell growth and adhesionbetween cells and anchors the actin cytoskeleton. In some instance,beta-catenin is responsible for transmitting the contact inhibitionsignal that causes the cells to stop dividing once the epithelial sheetis complete. Beta-catenin is also a key nuclear effector of the Wntsignaling pathway. In some instances, imbalance in the structural andsignaling properties of beta-catenin results in diseases and deregulatedgrowth connected to malignancies such as cancer. For example,overexpression of beta-catenin has been linked to cancers such asgastric cancer (Suriano, et al., “Beta-catenin (CTNNB1) geneamplification: a new mechanism of protein overexpression in cancer,”Genes Chromosomes Cancer 42(3): 238-246 (2005)). In some cases,mutations in CTNNB1 gene have been linked to cancer development (e.g.,colon cancer, melanoma, hepatocellular carcinoma, ovarian cancer,endometrial cancer, medulloblastoma pilomatricomas, or prostratecancer), and in some instances, has been linked to metastaticprogression. In additional cases, mutations in the CTNNB1 gene causebeta-catenin to translocate to the nucleus without any external stimulusand drive the transcription of its target genes continuously. In somecases, the potential of beta-catenin to change the previously epithelialphenotype of affected cells into an invasive, mesenchyme-like typecontributes to metastasis formation.

In some embodiments, CTNNB1 gene is wild type CTNNB1 or CTNNB1comprising one or more mutations. In some instances, CTNNB1 is wild typeCTNNB1. In some instances, CTNNB1 is CTNNB1 comprising one or moremutations. In some instances, the polynucleic acid molecule is apolynucleic acid molecule that hybridizes to a target region of wildtype CTNNB1. In some instances, the polynucleic acid molecule is apolynucleic acid molecule that hybridizes to a target region of CTNNB1comprising a mutation (e.g., a substitution, a deletion, or anaddition).

In some embodiments, CTNNB1 DNA or RNA comprises one or more mutations.In some embodiments, CTNNB1 DNA or RNA comprises one or more mutationswithin one or more exons. In some instances, the one or more exonscomprise exon 3. In some instances, CTNNB1 DNA or RNA comprises one ormore mutations at codons 32, 33, 34, 37, 41, 45, 183, 245, 287 or acombination thereof. In some instances, CTNNB1 DNA or RNA comprises oneor more mutations at positions corresponding to amino acid residues 25,31, 32, 33, 34, 35, 36, 37, 41, 45, 140, 162, 170, 199, 213, 215, 257,303, 322, 334, 354, 367, 373, 383, 387, 402, 426, 453, 474, 486, 515,517, 535, 553, 555, 582, 587, 619, 623, 641, 646, 688, 703, 710, 712,714, 724, 738, 777, or a combination thereof of the CTNNB1 polypeptide.In some embodiments, CTNNB1 DNA or RNA comprises one or more mutationsat positions corresponding to amino acid residues selected from W25(nonsense mutation), L31M, D32A, D32N, D32Y, D32G, D32H, S33C, S33Y,S33F, S33P, G34R, G34E, G34V, I35S, H36Y, S37F, S37P, S37C, S37A, T41N,T41A, T41I, S45Y, S45F, S45C, I140T, D162E, K170M, V199I, C213F, A215T,T257I, I303M, Q322K, E334K, K354T, G367V, P373S, W383G, N387K, L402F,N426D, R453L, R453Q, 8474 (nonsense mutation), R486C, R515Q, L517F, R535(nonsense mutation), R535Q, M553V, G555A, R582Q, R587Q, C619Y, Q623E,T641 (frame shift), S646F, M688T, Q703H, R710H, D712N, P714R, Y724H,E738K, F777S, or a combination thereof of the CTNNB1 polypeptide.

In some embodiments, the polynucleic acid molecule hybridizes to atarget region of CTNNB1 DNA or RNA comprising one or more mutations. Insome embodiments, the polynucleic acid molecule hybridizes to a targetregion of CTNNB1 DNA or RNA comprising one or more mutations within exon3. In some embodiments, the polynucleic acid molecule hybridizes to atarget region of CTNNB1 DNA or RNA comprising one or more mutations atcodons 32, 33, 34, 37, 41, 45, 183, 245, 287 or a combination thereof.In some embodiments, the polynucleic acid molecule hybridizes to atarget region of CTNNB1 DNA or RNA comprising one or more mutations atpositions corresponding to amino acid residues 25, 31, 32, 33, 34, 35,36, 37, 41, 45, 140, 162, 170, 199, 213, 215, 257, 303, 322, 334, 354,367, 373, 383, 387, 402, 426, 453, 474, 486, 515, 517, 535, 553, 555,582, 587, 619, 623, 641, 646, 688, 703, 710, 712, 714, 724, 738, 777, ora combination thereof of the CTNNB1 polypeptide. In some embodiments,the polynucleic acid molecule hybridizes to a target region of CTNNB1DNA or RNA comprising one or more mutations selected from W25 (nonsensemutation), L31M, D32A, D32N, D32Y, D32G, D32H, S33C, S33Y, S33F, S33P,G34R, G34E, G34V, I35S, H36Y, S37F, S37P, S37C, S37A, T41N, T41A, T41I,S45Y, S45F, S45C, I140T, D162E, K170M, V199I, C213F, A215T, T257I,I303M, Q322K, E334K, K354T, G367V, P373S, W383G, N387K, L402F, N426D,R453L, R453Q, R474 (nonsense mutation), R486C, R515Q, L517F, R535(nonsense mutation), R535Q, M553V, G555A, R582Q, R587Q, C619Y, Q623E,T641 (frame shift), S646F, M688T, Q703H, R710H, D712N, P714R, Y724H,E738K, F777S, or a combination thereof of the CTNNB1 polypeptide.

In some embodiments, beta-catenin associated genes further comprisePIK3CA, PIK3CB, and MYC. In some embodiments, beta-catenin associatedgenes further comprise PIK3CA DNA or RNA. PIK3CA(phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alphaor p110α protein) is a class i PI 3-kinase catalytic subunit that usesATP to phosphorylate phosphatidylinositols. In some embodiments, PIK3CAgene is wild type PIK3CA or PIK3CA comprising one or more mutations. Insome instances, PIK3CA DNA or RNA is wild type PIK3CA. In someinstances, PIK3CA DNA or RNA comprises one or more mutations. In someinstances, the polynucleic acid molecule hybridizes to a target regionof wild type PIK3CA DNA or RNA. In some instances, the polynucleic acidmolecule hybridizes to a target region of PIK3CA DNA or RNA comprising amutation (e.g., a substitution, a deletion, or an addition).

In some embodiments, PIK3CA DNA or RNA comprises one or more mutations.In some embodiments, PIK3CA DNA or RNA comprises one or more mutationwithin one or more exons. In some instances, PIK3CA DNA or RNA comprisesone or more mutation within exons 9 and/or 20. In some instances, PIK3CADNA or RNA comprises one or more mutations at positions corresponding toamino acid residues 1, 4, 10-16, 11-18, 11, 12, 38, 39, 65, 72, 75, 79,81, 83, 88, 90, 93, 102, 103, 103-104, 103-106, 104, 105-108, 106,106-107, 106-108, 107, 108, 109-112, 110, 111, 113, 115, 137, 170, 258,272, 279, 320, 328, 335, 342, 344, 345, 350, 357, 359, 363, 364, 365,366, 378, 398, 401, 417, 420, 447-455, 449, 449-457, 451, 453, 454, 455,455-460, 463-465, 471, 495, 522, 538, 539, 542, 545, 546, 547, 576, 604,614, 617, 629, 643, 663, 682, 725, 726, 777, 791, 818, 866, 901, 909,939, 951, 958, 970, 971, 975, 992, 1004, 1007, 1016, 1017, 1021, 1025,1029, 1037, 1040, 1043, 1044, 1045, 1047, 1048, 1049, 1052, 1065, 1069,or a combination thereof of the PIK3CA polypeptide. In some embodiments,PIK3CA DNA or RNA comprises one or more mutations at positionscorresponding to amino acid residues selected from M1V, R4 (nonsensemutation), L10-M16 (deletion), W11-P18 (deletion), W11L, G12D, R38L,R38H, R38C, R38S, E39K, E39G, E65K, S72G, Q75E, R79M, E81K, E81(deletion), F83Y, R88Q, C90Y, C90G, R93Q, R93W, I102 (deletion), E103G,E103-P104 (deletion), E103-G106 (deletion), P104L, V105-R108 (deletion),G106V, G106-N107 (deletion), G106-R108 (deletion), G106R, N107S, R108L,R108H, E109-I112 (deletion), E110 (deletion), K111E, K111R, K111N, K111(deletion), L113 (deletion), R115L, Q137L, N170S, D258N, Y272 (nonsensemutation), L279I, G320V, W328S, R335G, T342S, V344G, V344M, V344A,N345K, N345I, N345T, D350N, D350G, R357Q, G359R, G363A, G364R, E365K,E365V, P366R, C378R, C378Y, R398H, R401Q, E417K, C420R, C420G, P447-L455(deletion), P449L, P449-N457 (deletion), G451R, G451V, E453K, E453Q,E453D, D454Y, L455 (frame shift insertion), L455-G460 (deletion),G463-N465 (deletion), P471L, P471A, H495L, H495Y, E522A, D538N, P539R,E542K, E542V, E542G, E542Q, E542A, E545K, E545A, E545G, E545Q, E545D,Q546K, Q546R, Q546P, E547D, S576Y, C604R, F614I, A617W, S629C, Q643H,I663S, Q682 (deletion), D725N, W726K, R777M, E791Q, R818C, L866W, C901F,F909L, D939G, R951C, Q958R, E970K, C971R, R975S, R992P, M1004I, G1007R,F1016C, D1017H, Y1021H, Y1021C, T1025A, T1025S, D1029H, E1037K, M1040V,M1043V, M1043I, N1044K, N1044Y, D1045V, H1047R, H1047L, H1047Y, H1047Q,H1048R, G1049R, T1052K, H1065L, I069W (nonstop mutation), or acombination thereof of the PIK3CA polypeptide.

In some embodiments, the polynucleic acid molecule hybridizes to atarget region of PIK3CA DNA or RNA comprising one or more mutations. Insome embodiments, the polynucleic acid molecule hybridizes to a targetregion of PIK3CA DNA or RNA comprising one or more mutations within anexon. In some embodiments, the polynucleic acid molecule hybridizes to atarget region of PIK3CA DNA or RNA comprising one or more mutationswithin exon 9 or exon 20. In some embodiments, the polynucleic acidmolecule hybridizes to a target region of PIK3CA DNA or RNA comprisingone or more mutations at positions corresponding to amino acid residues1, 4, 10-16, 11-18, 11, 12, 38, 39, 65, 72, 75, 79, 81, 83, 88, 90, 93,102, 103, 103-104, 103-106, 104, 105-108, 106, 106-107, 106-108, 107,108, 109-112, 110, 111, 113, 115, 137, 170, 258, 272, 279, 320, 328,335, 342, 344, 345, 350, 357, 359, 363, 364, 365, 366, 378, 398, 401,417, 420, 447-455, 449, 449-457, 451, 453, 454, 455, 455-460, 463-465,471, 495, 522, 538, 539, 542, 545, 546, 547, 576, 604, 614, 617, 629,643, 663, 682, 725, 726, 777, 791, 818, 866, 901, 909, 939, 951, 958,970, 971, 975, 992, 1004, 1007, 1016, 1017, 1021, 1025, 1029, 1037,1040, 1043, 1044, 1045, 1047, 1048, 1049, 1052, 1065, 1069, or acombination thereof of the PIK3CA polypeptide. In some embodiments, thepolynucleic acid molecule is a polynucleic acid molecule that hybridizesto a target region of PIK3CA DNA or RNA comprising one or more mutationsat positions corresponding to amino acid residues selected from M1V, R4(nonsense mutation), L10-M16 (deletion), W11-P18 (deletion), W11L, G12D,R38L, R38H, R38C, R38S, E39K, E39G, E65K, S72G, Q75E, R79M, E81K, E81(deletion), F83Y, R88Q, C90Y, C90G, R93Q, R93W, I102 (deletion), E103G,E103-P104 (deletion), E103-G106 (deletion), P104L, V105-R108 (deletion),G106V, G106-N107 (deletion), G106-R108 (deletion), G106R, N107S, R108L,R108H, E109-I112 (deletion), E110 (deletion), K111E, K111R, K111N, K111(deletion), L113 (deletion), R115L, Q137L, N170S, D258N, Y272 (nonsensemutation), L279I, G320V, W328S, R335G, T342S, V344G, V344M, V344A,N345K, N345I, N345T, D350N, D350G, R357Q, G359R, G363A, G364R, E365K,E365V, P366R, C378R, C378Y, R398H, R401Q, E417K, C420R, C420G, P447-L455(deletion), P449L, P449-N457 (deletion), G451R, G451V, E453K, E453Q,E453D, D454Y, L455 (frame shift insertion), L455-G460 (deletion),G463-N465 (deletion), P471L, P471A, H495L, H495Y, E522A, D538N, P539R,E542K, E542V, E542G, E542Q, E542A, E545K, E545A, E545G, E545Q, E545D,Q546K, Q546R, Q546P, E547D, S576Y, C604R, F614I, A617W, S629C, Q643H,I663S, Q682 (deletion), D725N, W726K, R777M, E791Q, R818C, L866W, C901F,F909L, D939G, R951C, Q958R, E970K, C971R, R975S, R992P, M1004I, G1007R,F1016C, D1017H, Y1021H, Y1021C, T1025A, T1025S, D1029H, E1037K, M1040V,M1043V, M1043I, N1044K, N1044Y, D1045V, H1047R, H1047L, H1047Y, H1047Q,H1048R, G1049R, T1052K, H1065L, 1069W (nonstop mutation), or acombination thereof of the PIK3CB polypeptide.

In some embodiments, beta-catenin associated genes further comprisePIK3CB. In some embodiments, PIK3CB gene is wild type or comprises oneor more mutations. In some instances, PIK3CB DNA or RNA is wild typePIK3CB DNA or RNA. In some instances, PIK3CB DNA or RNA comprises one ormore mutations. In some instances, the polynucleic acid moleculehybridizes to a target region of wild type PIK3CB DNA or RNA. In someinstances, the polynucleic acid molecule hybridizes to a target regionof PIK3CB DNA or RNA comprising a mutation (e.g., a substitution, adeletion, or an addition).

In some embodiments, PIK3CB DNA or RNA comprises one or more mutations.In some embodiments, PIK3CB DNA or RNA comprises one or more mutationswithin one or more exons. In some instances, PIK3CB DNA or RNA comprisesone or more mutations at positions corresponding to amino acid residues18, 19, 21, 28, 50, 61, 68, 103, 135, 140, 167, 252, 270, 290, 301, 304,321, 369, 417, 442, 470, 497, 507, 512, 540, 551, 552, 554, 562, 567,593, 595, 619, 628, 668, 768, 805, 824, 830, 887, 967, 992, 1005, 1020,1036, 1046, 1047, 1048, 1049, 1051, 1055, 1067, or a combination thereofof the PIK3CB polypeptide. In some embodiments, PIK3CB DNA or RNAcomprises one or more mutations at positions corresponding to amino acidresidues selected from W18 (nonsense mutation), A19V, D21H, G28S, A50P,K61T, M68I, R103K, H135N, L140S, S167C, G252W, R270W, K290N, E301V,I304R, R321Q, V369I, T417M, N442K, E470K, E497D, P507S, I512M, E540(nonsense mutation), C551R, E552K, E554K, R562 (nonsense mutation),E567D, A593V, L595P, V619A, R628 (nonsense mutation), R668W, L768F,K805E, D824E, A830T, E887 (nonsense mutation), V967A, I992T, A1005V,D1020H, E1036K, D1046N, E1047K, A1048V, L1049R, E1051K, T1055A, D1067V,D1067A, or a combination thereof of the PIK3CB polypeptide.

In some embodiments, the polynucleic acid molecule hybridizes to atarget region of PIK3CB DNA or RNA comprising one or more mutations. Insome embodiments, the polynucleic acid molecule hybridizes to a targetregion of PIK3CB DNA or RNA comprising one or more mutations within anexon. In some embodiments, the polynucleic acid molecule hybridizes to atarget region of PIK3CB DNA or RNA comprising one or more mutations atpositions corresponding to amino acid residues 18, 19, 21, 28, 50, 61,68, 103, 135, 140, 167, 252, 270, 290, 301, 304, 321, 369, 417, 442,470, 497, 507, 512, 540, 551, 552, 554, 562, 567, 593, 595, 619, 628,668, 768, 805, 824, 830, 887, 967, 992, 1005, 1020, 1036, 1046, 1047,1048, 1049, 1051, 1055, 1067, or a combination thereof of the PIK3CBpolypeptide. In some embodiments, the polynucleic acid moleculehybridizes to a target region of PIK3CB DNA or RNA comprising one ormore mutations at positions corresponding to amino acid residuesselected from W18 (nonsense mutation), A19V, D21H, G28S, A50P, K61T,M68I, R103K, H135N, L140S, S167C, G252W, R270W, K290N, E301V, I304R,R321Q, V369I, T417M, N442K, E470K, E497D, P507S, I512M, E540 (nonsensemutation), C551R, E552K, E554K, R562 (nonsense mutation), E567D, A593V,L595P, V619A, R628 (nonsense mutation), R668W, L768F, K805E, D824E,A830T, E887 (nonsense mutation), V967A, I992T, A1005V, D1020H, E1036K,D1046N, E1047K, A1048V, L1049R, E1051K, T1055A, D1067V, D1067A, or acombination thereof of the PIK3CB polypeptide.

In some embodiments, beta-catenin associated genes further comprise MYC.In some embodiments, MYC gene is wild type MYC or MYC comprising one ormore mutations. In some instances, MYC is wild type MYC DNA or RNA. Insome instances, MYC DNA or RNA comprises one or more mutations. In someinstances, the polynucleic acid molecule hybridizes to a target regionof wild type MYC DNA or RNA. In some instances, the polynucleic acidmolecule is a polynucleic acid molecule that hybridizes to a targetregion of MYC DNA or RNA comprising a mutation (e.g., a substitution, adeletion, or an addition).

In some embodiments, MYC DNA or RNA comprises one or more mutations. Insome embodiments, MYC DNA or RNA comprises one or more mutation withinone or more exons. In some instances, MYC DNA or RNA comprises one ormore mutations within exon 2 or exon 3. In some instances, MYC DNA orRNA comprises one or more mutations at positions corresponding to aminoacid residues 2, 7, 17, 20, 32, 44, 58, 59, 76, 115, 138, 141, 145, 146,169, 175, 188, 200, 202, 203, 248, 251, 298, 321, 340, 369, 373, 374,389, 395, 404, 419, 431, 439, or a combination thereof. In someembodiments, MYC DNA or RNA comprises one or more mutations at positionscorresponding to amino acid residues selected from P2L, F7L, D17N, Q20E,Y32N, A44V, A44T, T58I, P59L, A76V, F115L, F138S, A141S, V145I, S146L,S169C, S175N, C188F, N200S, S202N, S203T, T248S, D251E, S298Y, Q321E,V340D, V369D, T373K, H374R, F389L, Q395H, K404N, L419M, E431K, R439Q, ora combination thereof of the MYC polypeptide.

In some embodiments, the polynucleic acid molecule hybridizes to atarget region of MYC DNA or RNA comprising one or more mutations. Insome embodiments, the polynucleic acid molecule hybridizes to a targetregion of MYC DNA or RNA comprising one or more mutations within anexon. In some embodiments, the polynucleic acid molecule hybridizes to atarget region of MYC DNA or RNA comprising one or more mutations withinexon 2 or exon 3. In some embodiments, the polynucleic acid moleculehybridizes to a target region of MYC DNA or RNA comprising one or moremutations at positions corresponding to amino acid residues 2, 7, 17,20, 32, 44, 58, 59, 76, 115, 138, 141, 145, 146, 169, 175, 188, 200,202, 203, 248, 251, 298, 321, 340, 369, 373, 374, 389, 395, 404, 419,431, 439, or a combination thereof of the MYC polypeptide. In someembodiments, the polynucleic acid molecule hybridizes to a target regionof MYC DNA or RNA comprising one or more mutations at positionscorresponding to amino acid residues selected from P2L, F7L, D17N, Q20E,Y32N, A44V, A44T, T58I, P59L, A76V, F115L, F138S, A141S, V145I, S146L,S169C, S175N, C188F, N200S, S202N, S203T, T248S, D251E, S298Y, Q321E,V340D, V369D, T373K, H374R, F389L, Q395H, K404N, L419M, E431K, R439Q, ora combination thereof of the MYC polypeptide.

Polynucleic Acid Molecules that Target HypoxanthinePhosphoribosyltransferase 1 (HPRT1)

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a transferasethat catalyzes the conversion of hypoxanthine to inosine monophosphateand guanine to guanosine monophosphate. HGPRT is encoded by thehypoxanthine Phosphoribosyltransferase 1 (HPRT1) gene.

In some embodiments, HPRT1 DNA or RNA is wild type or comprises one ormore mutations. In some instances, HPRT1 DNA or RNA comprises one ormore mutations within one or more exons. In some instances, the one ormore exons comprise exon 2, exon 3, exon 4, exon 6, exon 8, or exon 9.In some instances, HPRT1 DNA or RNA comprises one or more mutations atpositions corresponding to amino acid residues 35, 48, 56, 74, 87, 129,154, 162, 195, 200, 210, or a combination thereof of the HPRT1polypeptide. In some embodiments, the polynucleic acid moleculehybridizes to a target region of HPRT1 DNA or RNA comprising one or moremutations selected from V35M, R48H, E56D, F74L, R87I, N129 (splice-sitemutation), N154H, S162 (splice-site mutation), Y195C, Y195N, R200M,E210K, or a combination thereof of the HPRT1 polypeptide.

In some embodiments, the polynucleic acid molecule hybridizes to atarget region of HPRT1 DNA or RNA comprising one or more mutations. Insome embodiments, the polynucleic acid molecule hybridizes to a targetregion of HPRT1 DNA or RNA comprising one or more mutations within exon2, exon 3, exon 4, exon 6, exon 8, or exon 9. In some embodiments, thepolynucleic acid molecule hybridizes to a target region of HPRT1 DNA orRNA comprising one or more mutations at positions corresponding to aminoacid residues 35, 48, 56, 74, 87, 129, 154, 162, 195, 200, 210, or acombination thereof of the HPRT1 polypeptide. In some embodiments, thepolynucleic acid molecule hybridizes to a target region of HPRT1 DNA orRNA comprising one or more mutations selected from V35M, R48H, E56D,F74L, R87I, N129 (splice-site mutation), N154H, S162 (splice-sitemutation), Y195C, Y195N, R200M, E210K, or a combination thereof of theHPRT1 polypeptide.

Polynucleic Acid Molecule Sequences

In some embodiments, the polynucleic acid molecule comprises a sequencethat hybridizes to a target sequence illustrated in Tables 1, 4, 7, 8,or 10. In some instances, the polynucleic acid molecule is B. In someinstances, the polynucleic acid molecule B comprises a sequence thathybridizes to a target sequence illustrated in Table 1 (KRAS targetsequences). In some instances, the polynucleic acid molecule B comprisesa sequence that hybridizes to a target sequence illustrated in Table 4(EGFR target sequences). In some cases, the polynucleic acid molecule Bcomprises a sequence that hybridizes to a target sequence illustrated inTable 7 (AR target sequences). In some cases, the polynucleic acidmolecule B comprises a sequence that hybridizes to a target sequenceillustrated in Table 8 (β-catenin target sequences). In additionalcases, the polynucleic acid molecule B comprises a sequence thathybridizes to a target sequence illustrated in Table 10 (PIK3CA andPIK3CB target sequences).

In some embodiments, the polynucleic acid molecule B comprises asequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence listedin Table 2 or Table 3. In some embodiments, the polynucleic acidmolecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identityto SEQ ID NOs: 16-75. In some embodiments, the polynucleic acid moleculecomprises a sequence having at least 50% sequence identity to SEQ IDNOs: 16-75. In some embodiments, the polynucleic acid molecule comprisesa sequence having at least 60% sequence identity to SEQ ID NOs: 16-75.In some embodiments, the polynucleic acid molecule comprises a sequencehaving at least 70% sequence identity to SEQ ID NOs: 16-75. In someembodiments, the polynucleic acid molecule comprises a sequence havingat least 75% sequence identity to SEQ ID NOs: 16-75. In someembodiments, the polynucleic acid molecule comprises a sequence havingat least 80% sequence identity to SEQ ID NOs: 16-75. In someembodiments, the polynucleic acid molecule comprises a sequence havingat least 85% sequence identity to SEQ ID NOs: 16-75. In someembodiments, the polynucleic acid molecule comprises a sequence havingat least 90% sequence identity to SEQ ID NOs: 16-75. In someembodiments, the polynucleic acid molecule comprises a sequence havingat least 95% sequence identity to SEQ ID NOs: 16-75. In someembodiments, the polynucleic acid molecule comprises a sequence havingat least 96% sequence identity to SEQ ID NOs: 16-75. In someembodiments, the polynucleic acid molecule comprises a sequence havingat least 97% sequence identity to SEQ ID NOs: 16-75. In someembodiments, the polynucleic acid molecule comprises a sequence havingat least 98% sequence identity to SEQ ID NOs: 16-75. In someembodiments, the polynucleic acid molecule comprises a sequence havingat least 99% sequence identity to SEQ ID NOs: 16-75. In someembodiments, the polynucleic acid molecule consists of SEQ ID NOs:16-75.

In some embodiments, the polynucleic acid molecule B comprises a firstpolynucleotide and a second polynucleotide. In some instances, the firstpolynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to SEQ ID NOs: 16-75. In some cases, the second polynucleotidecomprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ IDNOs: 16-75. In some cases, the polynucleic acid molecule comprises afirst polynucleotide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ IDNOs: 16-75 and a second polynucleotide having at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to SEQ ID NOs: 16-75.

In some embodiments, the polynucleic acid molecule B comprises asequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence listedin Table 5 or Table 6. In some embodiments, the polynucleic acidmolecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identityto SEQ ID NOs: 452-1955. In some embodiments, the polynucleic acidmolecule comprises a sequence having at least 50% sequence identity toSEQ ID NOs: 452-1955. In some embodiments, the polynucleic acid moleculecomprises a sequence having at least 60% sequence identity to SEQ IDNOs: 452-1955. In some embodiments, the polynucleic acid moleculecomprises a sequence having at least 70% sequence identity to SEQ IDNOs: 452-1955. In some embodiments, the polynucleic acid moleculecomprises a sequence having at least 75% sequence identity to SEQ IDNOs: 452-1955. In some embodiments, the polynucleic acid moleculecomprises a sequence having at least 80% sequence identity to SEQ IDNOs: 452-1955. In some embodiments, the polynucleic acid moleculecomprises a sequence having at least 85% sequence identity to SEQ IDNOs: 452-1955. In some embodiments, the polynucleic acid moleculecomprises a sequence having at least 90% sequence identity to SEQ IDNOs: 452-1955. In some embodiments, the polynucleic acid moleculecomprises a sequence having at least 95% sequence identity to SEQ IDNOs: 452-1955. In some embodiments, the polynucleic acid moleculecomprises a sequence having at least 96% sequence identity to SEQ IDNOs: 452-1955. In some embodiments, the polynucleic acid moleculecomprises a sequence having at least 97% sequence identity to SEQ IDNOs: 452-1955. In some embodiments, the polynucleic acid moleculecomprises a sequence having at least 98% sequence identity to SEQ IDNOs: 452-1955. In some embodiments, the polynucleic acid moleculecomprises a sequence having at least 99% sequence identity to SEQ IDNOs: 452-1955. In some embodiments, the polynucleic acid moleculeconsists of SEQ ID NOs: 452-1955.

In some embodiments, the polynucleic acid molecule B comprises a firstpolynucleotide and a second polynucleotide. In some instances, the firstpolynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to SEQ ID NOs: 452-1955. In some cases, the secondpolynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to SEQ ID NOs: 452-1955. In some cases, the polynucleic acidmolecule comprises a first polynucleotide having at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to SEQ ID NOs: 452-1955 and a second polynucleotide having atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to SEQ ID NOs: 452-1955.

In some embodiments, the polynucleic acid molecule B comprises asequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence listedin Table 7. In some embodiments, the polynucleic acid molecule comprisesa sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs:1956-1962. In some embodiments, the polynucleic acid molecule comprisesa sequence having at least 50% sequence identity to SEQ ID NOs:1956-1962. In some embodiments, the polynucleic acid molecule comprisesa sequence having at least 60% sequence identity to SEQ ID NOs:1956-1962. In some embodiments, the polynucleic acid molecule comprisesa sequence having at least 70% sequence identity to SEQ ID NOs:1956-1962. In some embodiments, the polynucleic acid molecule comprisesa sequence having at least 75% sequence identity to SEQ ID NOs:1956-1962. In some embodiments, the polynucleic acid molecule comprisesa sequence having at least 80% sequence identity to SEQ ID NOs:1956-1962. In some embodiments, the polynucleic acid molecule comprisesa sequence having at least 85% sequence identity to SEQ ID NOs:1956-1962. In some embodiments, the polynucleic acid molecule comprisesa sequence having at least 90% sequence identity to SEQ ID NOs:1956-1962. In some embodiments, the polynucleic acid molecule comprisesa sequence having at least 95% sequence identity to SEQ ID NOs:1956-1962. In some embodiments, the polynucleic acid molecule comprisesa sequence having at least 96% sequence identity to SEQ ID NOs:1956-1962. In some embodiments, the polynucleic acid molecule comprisesa sequence having at least 97% sequence identity to SEQ ID NOs:1956-1962. In some embodiments, the polynucleic acid molecule comprisesa sequence having at least 98% sequence identity to SEQ ID NOs:1956-1962. In some embodiments, the polynucleic acid molecule comprisesa sequence having at least 99% sequence identity to SEQ ID NOs:1956-1962. In some embodiments, the polynucleic acid molecule consistsof SEQ ID NOs: 1956-1962.

In some embodiments, the polynucleic acid molecule B comprises a firstpolynucleotide and a second polynucleotide. In some instances, the firstpolynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to SEQ ID NOs: 1956-1962. In some cases, the secondpolynucleotide comprises a sequence that is complementary to a sequencehaving at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 1956-1962. Insome instances, the polynucleic acid molecule comprises a firstpolynucleotide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs:1956-1962, and a second polynucleotide that is complementary to asequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs:1956-1962.

In some embodiments, the polynucleic acid molecule B comprises asequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence listedin Table 9. In some embodiments, the polynucleic acid molecule comprisesa sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs:1967-2002. In some embodiments, the polynucleic acid molecule comprisesa sequence having at least 50% sequence identity to SEQ ID NOs:1967-2002. In some embodiments, the polynucleic acid molecule comprisesa sequence having at least 60% sequence identity to SEQ ID NOs:1967-2002. In some embodiments, the polynucleic acid molecule comprisesa sequence having at least 70% sequence identity to SEQ ID NOs:1967-2002. In some embodiments, the polynucleic acid molecule comprisesa sequence having at least 75% sequence identity to SEQ ID NOs:1967-2002. In some embodiments, the polynucleic acid molecule comprisesa sequence having at least 80% sequence identity to SEQ ID NOs:1967-2002. In some embodiments, the polynucleic acid molecule comprisesa sequence having at least 85% sequence identity to SEQ ID NOs:1967-2002. In some embodiments, the polynucleic acid molecule comprisesa sequence having at least 90% sequence identity to SEQ ID NOs:1967-2002. In some embodiments, the polynucleic acid molecule comprisesa sequence having at least 95% sequence identity to SEQ ID NOs:1967-2002. In some embodiments, the polynucleic acid molecule comprisesa sequence having at least 96% sequence identity to SEQ ID NOs:1967-2002. In some embodiments, the polynucleic acid molecule comprisesa sequence having at least 97% sequence identity to SEQ ID NOs:1967-2002. In some embodiments, the polynucleic acid molecule comprisesa sequence having at least 98% sequence identity to SEQ ID NOs:1967-2002. In some embodiments, the polynucleic acid molecule comprisesa sequence having at least 99% sequence identity to SEQ ID NOs:1967-2002. In some embodiments, the polynucleic acid molecule consistsof SEQ ID NOs: 1967-2002.

In some embodiments, the polynucleic acid molecule B comprises a firstpolynucleotide and a second polynucleotide. In some instances, the firstpolynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to SEQ ID NOs: 1967-2002. In some cases, the secondpolynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to SEQ ID NOs: 1967-2002. In some cases, the polynucleic acidmolecule comprises a first polynucleotide having at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to SEQ ID NOs: 1967-2002 and a second polynucleotide having atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to SEQ ID NOs: 1967-2002.

In some embodiments, the polynucleic acid molecule B comprises asequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence listedin Table 11. In some embodiments, the polynucleic acid moleculecomprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ IDNOs: 2013-2032. In some embodiments, the polynucleic acid moleculecomprises a sequence having at least 50% sequence identity to SEQ IDNOs: 2013-2032. In some embodiments, the polynucleic acid moleculecomprises a sequence having at least 60% sequence identity to SEQ IDNOs: 2013-2032. In some embodiments, the polynucleic acid moleculecomprises a sequence having at least 70% sequence identity to SEQ IDNOs: 2013-2032. In some embodiments, the polynucleic acid moleculecomprises a sequence having at least 75% sequence identity to SEQ IDNOs: 2013-2032. In some embodiments, the polynucleic acid moleculecomprises a sequence having at least 80% sequence identity to SEQ IDNOs: 2013-2032. In some embodiments, the polynucleic acid moleculecomprises a sequence having at least 85% sequence identity to SEQ IDNOs: 2013-2032. In some embodiments, the polynucleic acid moleculecomprises a sequence having at least 90% sequence identity to SEQ IDNOs: 2013-2032. In some embodiments, the polynucleic acid moleculecomprises a sequence having at least 95% sequence identity to SEQ IDNOs: 2013-2032. In some embodiments, the polynucleic acid moleculecomprises a sequence having at least 96% sequence identity to SEQ IDNOs: 2013-2032. In some embodiments, the polynucleic acid moleculecomprises a sequence having at least 97% sequence identity to SEQ IDNOs: 2013-2032. In some embodiments, the polynucleic acid moleculecomprises a sequence having at least 98% sequence identity to SEQ IDNOs: 2013-2032. In some embodiments, the polynucleic acid moleculecomprises a sequence having at least 99% sequence identity to SEQ IDNOs: 2013-2032. In some embodiments, the polynucleic acid moleculeconsists of SEQ ID NOs: 2013-2032.

In some embodiments, the polynucleic acid molecule B comprises a firstpolynucleotide and a second polynucleotide. In some instances, the firstpolynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to SEQ ID NOs: 2013-2032. In some cases, the secondpolynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to SEQ ID NOs: 2013-2032. In some cases, the polynucleic acidmolecule comprises a first polynucleotide having at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to SEQ ID NOs: 2013-2032 and a second polynucleotide having atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to SEQ ID NOs: 2013-2032.

In some embodiments, the polynucleic acid molecule B comprises asequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence listedin Table 12.

In some embodiments, the polynucleic acid molecule comprises a sequencehaving at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2082-2109 or2117. In some embodiments, the polynucleic acid molecule comprises asequence having at least 50% sequence identity to SEQ ID NOs: 2082-2109or 2117. In some embodiments, the polynucleic acid molecule comprises asequence having at least 60% sequence identity to SEQ ID NOs: 2082-2109or 2117. In some embodiments, the polynucleic acid molecule comprises asequence having at least 70% sequence identity to SEQ ID NOs: 2082-2109or 2117. In some embodiments, the polynucleic acid molecule comprises asequence having at least 75% sequence identity to SEQ ID NOs: 2082-2109or 2117. In some embodiments, the polynucleic acid molecule comprises asequence having at least 80% sequence identity to SEQ ID NOs: 2082-2109or 2117. In some embodiments, the polynucleic acid molecule comprises asequence having at least 85% sequence identity to SEQ ID NOs: 2082-2109or 2117. In some embodiments, the polynucleic acid molecule comprises asequence having at least 90% sequence identity to SEQ ID NOs: 2082-2109or 2117. In some embodiments, the polynucleic acid molecule comprises asequence having at least 95% sequence identity to SEQ ID NOs: 2082-2109or 2117. In some embodiments, the polynucleic acid molecule comprises asequence having at least 96% sequence identity to SEQ ID NOs: 2082-2109or 2117. In some embodiments, the polynucleic acid molecule comprises asequence having at least 97% sequence identity to SEQ ID NOs: 2082-2109or 2117. In some embodiments, the polynucleic acid molecule comprises asequence having at least 98% sequence identity to SEQ ID NOs: 2082-2109or 2117. In some embodiments, the polynucleic acid molecule comprises asequence having at least 99% sequence identity to SEQ ID NOs: 2082-2109or 2117. In some embodiments, the polynucleic acid molecule consists ofSEQ ID NOs: 2082-2109 or 2117.

In some embodiments, the polynucleic acid molecule B comprises a firstpolynucleotide and a second polynucleotide. In some instances, the firstpolynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to SEQ ID NOs: 2082-2109 or 2117. In some cases, the secondpolynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to SEQ ID NOs: 2082-2109 or 2117. In some cases, thepolynucleic acid molecule comprises a first polynucleotide having atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to SEQ ID NOs: 2082-2109 or 2117 and asecond polynucleotide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ IDNOs: 2082-2109 or 2117.

Polynucleic Acid Molecules

In some embodiments, the polynucleic acid molecule described hereincomprises RNA or DNA. In some cases, the polynucleic acid moleculecomprises RNA. In some instances, RNA comprises short interfering RNA(siRNA), short hairpin RNA (shRNA), microRNA (miRNA), double-strandedRNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), or heterogeneousnuclear RNA (hnRNA). In some instances, RNA comprises shRNA. In someinstances, RNA comprises miRNA. In some instances, RNA comprises dsRNA.In some instances, RNA comprises tRNA. In some instances, RNA comprisesrRNA. In some instances, RNA comprises hnRNA. In some instances, the RNAcomprises siRNA. In some instances, the polynucleic acid moleculecomprises siRNA. In some cases, B comprises siRNA.

In some embodiments, the polynucleic acid molecule is from about 10 toabout 50 nucleotides in length. In some instances, the polynucleic acidmolecule is from about 10 to about 30, from about 15 to about 30, fromabout 18 to about 25, from about 18 to about 24, from about 19 to about23, or from about 20 to about 22 nucleotides in length.

In some embodiments, the polynucleic acid molecule is about 50nucleotides in length. In some instances, the polynucleic acid moleculeis about 45 nucleotides in length. In some instances, the polynucleicacid molecule is about 40 nucleotides in length. In some instances, thepolynucleic acid molecule is about 35 nucleotides in length. In someinstances, the polynucleic acid molecule is about 30 nucleotides inlength. In some instances, the polynucleic acid molecule is about 25nucleotides in length. In some instances, the polynucleic acid moleculeis about 20 nucleotides in length. In some instances, the polynucleicacid molecule is about 19 nucleotides in length. In some instances, thepolynucleic acid molecule is about 18 nucleotides in length. In someinstances, the polynucleic acid molecule is about 17 nucleotides inlength. In some instances, the polynucleic acid molecule is about 16nucleotides in length. In some instances, the polynucleic acid moleculeis about 15 nucleotides in length. In some instances, the polynucleicacid molecule is about 14 nucleotides in length. In some instances, thepolynucleic acid molecule is about 13 nucleotides in length. In someinstances, the polynucleic acid molecule is about 12 nucleotides inlength. In some instances, the polynucleic acid molecule is about 11nucleotides in length. In some instances, the polynucleic acid moleculeis about 10 nucleotides in length. In some instances, the polynucleicacid molecule is from about 10 to about 50 nucleotides in length. Insome instances, the polynucleic acid molecule is from about 10 to about45 nucleotides in length. In some instances, the polynucleic acidmolecule is from about 10 to about 40 nucleotides in length. In someinstances, the polynucleic acid molecule is from about 10 to about 35nucleotides in length. In some instances, the polynucleic acid moleculeis from about 10 to about 30 nucleotides in length. In some instances,the polynucleic acid molecule is from about 10 to about 25 nucleotidesin length. In some instances, the polynucleic acid molecule is fromabout 10 to about 20 nucleotides in length. In some instances, thepolynucleic acid molecule is from about 15 to about 25 nucleotides inlength. In some instances, the polynucleic acid molecule is from about15 to about 30 nucleotides in length. In some instances, the polynucleicacid molecule is from about 12 to about 30 nucleotides in length.

In some embodiments, the polynucleic acid molecule comprises a firstpolynucleotide. In some instances, the polynucleic acid moleculecomprises a second polynucleotide. In some instances, the polynucleicacid molecule comprises a first polynucleotide and a secondpolynucleotide. In some instances, the first polynucleotide is a sensestrand or passenger strand. In some instances, the second polynucleotideis an antisense strand or guide strand.

In some embodiments, the polynucleic acid molecule is a firstpolynucleotide. In some embodiments, the first polynucleotide is fromabout 10 to about 50 nucleotides in length. In some instances, the firstpolynucleotide is from about 10 to about 30, from about 15 to about 30,from about 18 to about 25, from about 18 to about 24, from about 19 toabout 23, or from about 20 to about 22 nucleotides in length.

In some instances, the first polynucleotide is about 50 nucleotides inlength. In some instances, the first polynucleotide is about 45nucleotides in length. In some instances, the first polynucleotide isabout 40 nucleotides in length. In some instances, the firstpolynucleotide is about 35 nucleotides in length. In some instances, thefirst polynucleotide is about 30 nucleotides in length. In someinstances, the first polynucleotide is about 25 nucleotides in length.In some instances, the first polynucleotide is about 20 nucleotides inlength. In some instances, the first polynucleotide is about 19nucleotides in length. In some instances, the first polynucleotide isabout 18 nucleotides in length. In some instances, the firstpolynucleotide is about 17 nucleotides in length. In some instances, thefirst polynucleotide is about 16 nucleotides in length. In someinstances, the first polynucleotide is about 15 nucleotides in length.In some instances, the first polynucleotide is about 14 nucleotides inlength. In some instances, the first polynucleotide is about 13nucleotides in length. In some instances, the first polynucleotide isabout 12 nucleotides in length. In some instances, the firstpolynucleotide is about 11 nucleotides in length. In some instances, thefirst polynucleotide is about 10 nucleotides in length. In someinstances, the first polynucleotide is from about 10 to about 50nucleotides in length. In some instances, the first polynucleotide isfrom about 10 to about 45 nucleotides in length. In some instances, thefirst polynucleotide is from about 10 to about 40 nucleotides in length.In some instances, the first polynucleotide is from about 10 to about 35nucleotides in length. In some instances, the first polynucleotide isfrom about 10 to about 30 nucleotides in length. In some instances, thefirst polynucleotide is from about 10 to about 25 nucleotides in length.In some instances, the first polynucleotide is from about 10 to about 20nucleotides in length. In some instances, the first polynucleotide isfrom about 15 to about 25 nucleotides in length. In some instances, thefirst polynucleotide is from about 15 to about 30 nucleotides in length.In some instances, the first polynucleotide is from about 12 to about 30nucleotides in length.

In some embodiments, the polynucleic acid molecule is a secondpolynucleotide. In some embodiments, the second polynucleotide is fromabout 10 to about 50 nucleotides in length. In some instances, thesecond polynucleotide is from about 10 to about 30, from about 15 toabout 30, from about 18 to about 25, from about 18 to about 24, fromabout 19 to about 23, or from about 20 to about 22 nucleotides inlength.

In some instances, the second polynucleotide is about 50 nucleotides inlength. In some instances, the second polynucleotide is about 45nucleotides in length. In some instances, the second polynucleotide isabout 40 nucleotides in length. In some instances, the secondpolynucleotide is about 35 nucleotides in length. In some instances, thesecond polynucleotide is about 30 nucleotides in length. In someinstances, the second polynucleotide is about 25 nucleotides in length.In some instances, the second polynucleotide is about 20 nucleotides inlength. In some instances, the second polynucleotide is about 19nucleotides in length. In some instances, the second polynucleotide isabout 18 nucleotides in length. In some instances, the secondpolynucleotide is about 17 nucleotides in length. In some instances, thesecond polynucleotide is about 16 nucleotides in length. In someinstances, the second polynucleotide is about 15 nucleotides in length.In some instances, the second polynucleotide is about 14 nucleotides inlength. In some instances, the second polynucleotide is about 13nucleotides in length. In some instances, the second polynucleotide isabout 12 nucleotides in length. In some instances, the secondpolynucleotide is about 11 nucleotides in length. In some instances, thesecond polynucleotide is about 10 nucleotides in length. In someinstances, the second polynucleotide is from about 10 to about 50nucleotides in length. In some instances, the second polynucleotide isfrom about 10 to about 45 nucleotides in length. In some instances, thesecond polynucleotide is from about 10 to about 40 nucleotides inlength. In some instances, the second polynucleotide is from about 10 toabout 35 nucleotides in length. In some instances, the secondpolynucleotide is from about 10 to about 30 nucleotides in length. Insome instances, the second polynucleotide is from about 10 to about 25nucleotides in length. In some instances, the second polynucleotide isfrom about 10 to about 20 nucleotides in length. In some instances, thesecond polynucleotide is from about 15 to about 25 nucleotides inlength. In some instances, the second polynucleotide is from about 15 toabout 30 nucleotides in length. In some instances, the secondpolynucleotide is from about 12 to about 30 nucleotides in length.

In some embodiments, the polynucleic acid molecule comprises a firstpolynucleotide and a second polynucleotide. In some instances, thepolynucleic acid molecule further comprises a blunt terminus, anoverhang, or a combination thereof. In some instances, the bluntterminus is a 5′ blunt terminus, a 3′ blunt terminus, or both. In somecases, the overhang is a 5′ overhang, 3′ overhang, or both. In somecases, the overhang comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-basepairing nucleotides. In some cases, the overhang comprises 1, 2, 3, 4,5, or 6 non-base pairing nucleotides. In some cases, the overhangcomprises 1, 2, 3, or 4 non-base pairing nucleotides. In some cases, theoverhang comprises 1 non-base pairing nucleotide. In some cases, theoverhang comprises 2 non-base pairing nucleotides. In some cases, theoverhang comprises 3 non-base pairing nucleotides. In some cases, theoverhang comprises 4 non-base pairing nucleotides.

In some embodiments, the sequence of the polynucleic acid molecule is atleast 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%,or 99.5% complementary to a target sequence described herein. In someembodiments, the sequence of the polynucleic acid molecule is at least50% complementary to a target sequence described herein. In someembodiments, the sequence of the polynucleic acid molecule is at least60% complementary to a target sequence described herein. In someembodiments, the sequence of the polynucleic acid molecule is at least70% complementary to a target sequence described herein. In someembodiments, the sequence of the polynucleic acid molecule is at least80% complementary to a target sequence described herein. In someembodiments, the sequence of the polynucleic acid molecule is at least90% complementary to a target sequence described herein. In someembodiments, the sequence of the polynucleic acid molecule is at least95% complementary to a target sequence described herein. In someembodiments, the sequence of the polynucleic acid molecule is at least99% complementary to a target sequence described herein. In someinstances, the sequence of the polynucleic acid molecule is 100%complementary to a target sequence described herein.

In some embodiments, the sequence of the polynucleic acid molecule has 5or less mismatches to a target sequence described herein. In someembodiments, the sequence of the polynucleic acid molecule has 4 or lessmismatches to a target sequence described herein. In some instances, thesequence of the polynucleic acid molecule may has 3 or less mismatchesto a target sequence described herein. In some cases, the sequence ofthe polynucleic acid molecule may has 2 or less mismatches to a targetsequence described herein. In some cases, the sequence of thepolynucleic acid molecule may has 1 or less mismatches to a targetsequence described herein.

In some embodiments, the specificity of the polynucleic acid moleculethat hybridizes to a target sequence described herein is a 95%, 98%,99%, 99.5%, or 100% sequence complementarity of the polynucleic acidmolecule to a target sequence. In some instances, the hybridization is ahigh stringent hybridization condition.

In some embodiments, the polynucleic acid molecule hybridizes to atleast 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or morecontiguous bases of a target sequence described herein. In someembodiments, the polynucleic acid molecule hybridizes to at least 8contiguous bases of a target sequence described herein. In someembodiments, the polynucleic acid molecule hybridizes to at least 9contiguous bases of a target sequence described herein. In someembodiments, the polynucleic acid molecule hybridizes to at least 10contiguous bases of a target sequence described herein. In someembodiments, the polynucleic acid molecule hybridizes to at least 11contiguous bases of a target sequence described herein. In someembodiments, the polynucleic acid molecule hybridizes to at least 12contiguous bases of a target sequence described herein. In someembodiments, the polynucleic acid molecule hybridizes to at least 13contiguous bases of a target sequence described herein. In someembodiments, the polynucleic acid molecule hybridizes to at least 14contiguous bases of a target sequence described herein. In someembodiments, the polynucleic acid molecule hybridizes to at least 15contiguous bases of a target sequence described herein. In someembodiments, the polynucleic acid molecule hybridizes to at least 16contiguous bases of a target sequence described herein. In someembodiments, the polynucleic acid molecule hybridizes to at least 17contiguous bases of a target sequence described herein. In someembodiments, the polynucleic acid molecule hybridizes to at least 18contiguous bases of a target sequence described herein. In someembodiments, the polynucleic acid molecule hybridizes to at least 19contiguous bases of a target sequence described herein. In someembodiments, the polynucleic acid molecule hybridizes to at least 20contiguous bases of a target sequence described herein.

In some embodiments, the polynucleic acid molecule has reducedoff-target effect. In some instances, “off-target” or “off-targeteffects” refer to any instance in which a polynucleic acid polymerdirected against a given target causes an unintended effect byinteracting either directly or indirectly with another mRNA sequence, aDNA sequence or a cellular protein or other moiety. In some instances,an “off-target effect” occurs when there is a simultaneous degradationof other transcripts due to partial homology or complementarity betweenthat other transcript and the sense and/or antisense strand of thepolynucleic acid molecule.

In some embodiments, the polynucleic acid molecule comprises natural,synthetic, or artificial nucleotide analogues or bases. In some cases,the polynucleic acid molecule comprises combinations of DNA, RNA and/ornucleotide analogues. In some instances, the synthetic or artificialnucleotide analogues or bases comprise modifications at one or more ofribose moiety, phosphate moiety, nucleoside moiety, or a combinationthereof.

In some embodiments, a nucleotide analogue or artificial nucleotide basedescribed above comprises a nucleic acid with a modification at a 2′hydroxyl group of the ribose moiety. In some instances, the modificationincludes an H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, wherein R isan alkyl moiety. Exemplary alkyl moiety includes, but is not limited to,halogens, sulfurs, thiols, thioethers, thioesters, amines (primary,secondary, or tertiary), amides, ethers, esters, alcohols and oxygen. Insome instances, the alkyl moiety further comprises a modification. Insome instances, the modification comprises an azo group, a keto group,an aldehyde group, a carboxyl group, a nitro group, a nitroso, group, anitrile group, a heterocycle (e.g., imidazole, hydrazino orhydroxylamino) group, an isocyanate or cyanate group, or a sulfurcontaining group (e.g., sulfoxide, sulfone, sulfide, or disulfide). Insome instances, the alkyl moiety further comprises a heterosubstitution. In some instances, the carbon of the heterocyclic group issubstituted by a nitrogen, oxygen or sulfur. In some instances, theheterocyclic substitution includes but is not limited to, morpholino,imidazole, and pyrrolidino.

In some instances, the modification at the 2′ hydroxyl group is a2′-O-methyl modification or a 2′-O-methoxyethyl (2′-O-MOE) modification.In some cases, the 2′-O-methyl modification adds a methyl group to the2′ hydroxyl group of the ribose moiety whereas the 2′O-methoxyethylmodification adds a methoxyethyl group to the 2′ hydroxyl group of theribose moiety. Exemplary chemical structures of a 2′-O-methylmodification of an adenosine molecule and 2′O-methoxyethyl modificationof an uridine are illustrated below.

In some instances, the modification at the 2′ hydroxyl group is a2′-O-aminopropyl modification in which an extended amine groupcomprising a propyl linker binds the amine group to the 2′ oxygen. Insome instances, this modification neutralizes the phosphate-derivedoverall negative charge of the oligonucleotide molecule by introducingone positive charge from the amine group per sugar and thereby improvescellular uptake properties due to its zwitterionic properties. Anexemplary chemical structure of a 2′-O-aminopropyl nucleosidephosphoramidite is illustrated below.

In some instances, the modification at the 2′ hydroxyl group is a lockedor bridged ribose modification (e.g., locked nucleic acid or LNA) inwhich the oxygen molecule bound at the 2′ carbon is linked to the 4′carbon by a methylene group, thus forming a2′-C,4′-C-oxy-methylene-linked bicyclic ribonucleotide monomer.Exemplary representations of the chemical structure of LNA areillustrated below. The representation shown to the left highlights thechemical connectivities of an LNA monomer. The representation shown tothe right highlights the locked 3′-endo (³E) conformation of thefuranose ring of an LNA monomer.

In some instances, the modification at the 2′ hydroxyl group comprisesethylene nucleic acids (ENA) such as for example 2′-4′-ethylene-bridgednucleic acid, which locks the sugar conformation into a C₃′-endo sugarpuckering conformation. ENA are part of the bridged nucleic acids classof modified nucleic acids that also comprises LNA. Exemplary chemicalstructures of the ENA and bridged nucleic acids are illustrated below.

In some embodiments, additional modifications at the 2′ hydroxyl groupinclude 2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or2′-O—N-methylacetamido (2′-O-NMA).

In some embodiments, a nucleotide analogue comprises a modified basesuch as, but not limited to, 5-propynyluridine, 5-propynylcytidine,6-methyladenine, 6-methylguanine, N, N,-dimethyladenine,2-propyladenine, 2-propylguanine, 2-aminoadenine, 1-methylinosine,3-methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotideshaving a modification at the 5 position, 5-(2-amino) propyl uridine,5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine,2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine,7-methylguanosine, 2, 2-dimethylguanosine, 5-methylaminoethyluridine,5-methyloxyuridine, deazanucleotides (such as 7-deaza-adenosine,6-azouridine, 6-azocytidine, or 6-azothymidine), 5-methyl-2-thiouridine,other thio bases (such as 2-thiouridine, 4-thiouridine, and2-thiocytidine), dihydrouridine, pseudouridine, queuosine, archaeosine,naphthyl and substituted naphthyl groups, any O- and N-alkylated purinesand pyrimidines (such as N6-methyladenosine,5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridine-4-one,or pyridine-2-one), phenyl and modified phenyl groups such asaminophenol or 2,4,6-trimethoxy benzene, modified cytosines that act asG-clamp nucleotides, 8-substituted adenines and guanines, 5-substituteduracils and thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides,carboxyalkylaminoalkyi nucleotides, and alkylcarbonylalkylatednucleotides. Modified nucleotides also include those nucleotides thatare modified with respect to the sugar moiety, as well as nucleotideshaving sugars or analogs thereof that are not ribosyl. For example, thesugar moieties, in some cases are or are based on, mannoses, arabinoses,glucopyranoses, galactopyranoses, 4′-thioribose, and other sugars,heterocycles, or carbocycles. The term nucleotide also includes what areknown in the art as universal bases. By way of example, universal basesinclude but are not limited to 3-nitropyrrole, 5-nitroindole, ornebularine.

In some embodiments, a nucleotide analogue further comprises amorpholino, a peptide nucleic acid (PNA), a methylphosphonatenucleotide, a thiolphosphonate nucleotide, a 2′-fluoroN3-P5′-phosphoramidite, or a 1′, 5′-anhydrohexitol nucleic acid (HNA).Morpholino or phosphorodiamidate morpholino oligo (PMO) comprisessynthetic molecules whose structure mimics natural nucleic acidstructure but deviates from the normal sugar and phosphate structures.In some instances, the five member ribose ring is substituted with a sixmember morpholino ring containing four carbons, one nitrogen, and oneoxygen. In some cases, the ribose monomers are linked by aphosphordiamidate group instead of a phosphate group. In such cases, thebackbone alterations remove all positive and negative charges makingmorpholinos neutral molecules capable of crossing cellular membraneswithout the aid of cellular delivery agents such as those used bycharged oligonucleotides.

In some embodiments, a morpholino or PMO described above is a PMOcomprising a positive or cationic charge. In some instances, the PMO isPMOplus (Sarepta). PMOplus refers to phosphorodiamidate morpholinooligomers comprising any number of (1-piperazino)phosphinylideneoxy,(1-(4-(omega-guanidino-alkanoyl))-piperazino)phosphinylideneoxy linkages(e.g., as such those described in PCT Publication No. WO2008/036127. Insome cases, the PMO is a PMO described in U.S. Pat. No. 7,943,762.

In some embodiments, a morpholino or PMO described above is a PMO-X(Sarepta). In some cases, PMO-X refers to phosphorodiamidate morpholinooligomers comprising at least one linkage or at least one of thedisclosed terminal modifications, such as those disclosed in PCTPublication No. WO2011/150408 and U.S. Publication No. 2012/0065169.

In some embodiments, a morpholino or PMO described above is a PMO asdescribed in Table 5 of U.S. Publication No. 2014/0296321.

In some embodiments, peptide nucleic acid (PNA) does not contain sugarring or phosphate linkage and the bases are attached and appropriatelyspaced by oligoglycine-like molecules, therefore, eliminating a backbonecharge.

In some embodiments, one or more modifications optionally occur at theinternucleotide linkage. In some instances, modified internucleotidelinkage includes, but is not limited to, phosphorothioates;phosphorodithioates; methylphosphonates; 5′-alkylenephosphonates;5′-methylphosphonate; 3′-alkylene phosphonates; borontrifluoridates;borano phosphate esters and selenophosphates of 3′-5′linkage or2′-5′linkage; phosphotriesters; thionoalkylphosphotriesters; hydrogenphosphonate linkages; alkyl phosphonates; alkylphosphonothioates;arylphosphonothioates; phosphoroselenoates; phosphorodiselenoates;phosphinates; phosphoramidates; 3′-alkylphosphoramidates;aminoalkylphosphoramidates; thionophosphoramidates;phosphoropiperazidates; phosphoroanilothioates; phosphoroanilidates;ketones; sulfones; sulfonamides; carbonates; carbamates;methylenehydrazos; methylenedimethylhydrazos; formacetals;thioformacetals; oximes; methyleneiminos; methylenemethyliminos;thioamidates; linkages with riboacetyl groups; aminoethyl glycine; silylor siloxane linkages; alkyl or cycloalkyl linkages with or withoutheteroatoms of, for example, 1 to 10 carbons that are saturated orunsaturated and/or substituted and/or contain heteroatoms; linkages withmorpholino structures, amides, or polyamides wherein the bases areattached to the aza nitrogens of the backbone directly or indirectly;and combinations thereof.

In some instances, the modification is a methyl or thiol modificationsuch as methylphosphonate or thiolphosphonate modification. Exemplarythiolphosphonate nucleotide (left) and methylphosphonate nucleotide(right) are illustrated below.

In some instances, a modified nucleotide includes, but is not limitedto, 2′-fluoro N3-P5′-phosphoramidites illustrated as:

In some instances, a modified nucleotide includes, but is not limitedto, hexitol nucleic acid (or 1′, 5′-anhydrohexitol nucleic acids (HNA))illustrated as:

In some embodiments, one or more modifications comprise a modifiedphosphate backbone in which the modification generates a neutral oruncharged backbone. In some instances, the phosphate backbone ismodified by alkylation to generate an uncharged or neutral phosphatebackbone. As used herein, alkylation includes methylation, ethylation,and propylation. In some cases, an alkyl group, as used herein in thecontext of alkylation, refers to a linear or branched saturatedhydrocarbon group containing from 1 to 6 carbon atoms. In someinstances, exemplary alkyl groups include, but are not limited to,methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,tert-butyl, n-pentyl, isopentyl, neopentyl, hexyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3.3-dimethylbutyl, and 2-ethylbutylgroups. In some cases, a modified phosphate is a phosphate group asdescribed in U.S. Pat. No. 9,481,905.

In some embodiments, additional modified phosphate backbones comprisemethylphosphonate, ethylphosphonate, methylthiophosphonate, ormethoxyphosphonate. In some cases, the modified phosphate ismethylphosphonate. In some cases, the modified phosphate isethylphosphonate. In some cases, the modified phosphate ismethylthiophosphonate. In some cases, the modified phosphate ismethoxyphosphonate.

In some embodiments, one or more modifications further optionallyinclude modifications of the ribose moiety, phosphate backbone and thenucleoside, or modifications of the nucleotide analogues at the 3′ orthe 5′ terminus. For example, the 3′ terminus optionally include a 3′cationic group, or by inverting the nucleoside at the 3′-terminus with a3′-3′ linkage. In another alternative, the 3′-terminus is optionallyconjugated with an aminoalkyl group, e.g., a 3′ C5-aminoalkyl dT. In anadditional alternative, the 3′-terminus is optionally conjugated with anabasic site, e.g., with an apurinic or apyrimidinic site. In someinstances, the 5′-terminus is conjugated with an aminoalkyl group, e.g.,a 5′-O-alkylamino substituent. In some cases, the 5′-terminus isconjugated with an abasic site, e.g., with an apurinic or apyrimidinicsite.

In some embodiments, the polynucleic acid molecule comprises one or moreof the artificial nucleotide analogues described herein. In someinstances, the polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more of theartificial nucleotide analogues described herein. In some embodiments,the artificial nucleotide analogues include 2′-O-methyl,2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy,T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido(2′-O-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonatenucleotides, thiolphosphonate nucleotides, 2′-fluoroN3-P5′-phosphoramidites, or a combination thereof. In some instances,the polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more of the artificialnucleotide analogues selected from 2′-O-methyl, 2′-O-methoxyethyl(2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro,2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-DMAP), T-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O-NMA) modified, LNA, ENA,PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonatenucleotides, 2′-fluoro N3-P5′-phosphoramidites, or a combinationthereof. In some instances, the polynucleic acid molecule comprises 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, ormore of 2′-O-methyl modified nucleotides. In some instances, thepolynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 20, 25, or more of 2′-O-methoxyethyl(2′-O-MOE) modified nucleotides. In some instances, the polynucleic acidmolecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 20, 25, or more of thiolphosphonate nucleotides.

In some embodiments, the polynucleic acid molecule comprises at leastabout 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8,about 9, about 10, about 11, about 12, about 13, about 14, about 15,about 16, about 17, about 18, about 19, about 20, about 21, about 22, ormore modifications. In some instances, the polynucleic acid molecule isa polynucleic acid molecule of SEQ ID NOs: 16-75, 452-1955, 1956-1962,1967-2002, 2013-2032, 2082-2109, or 2117.

In some instances, the polynucleic acid molecule comprises at leastabout 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8,about 9, about 10, about 11, about 12, about 13, about 14, about 15,about 16, about 17, about 18, about 19, about 20, about 21, about 22, ormore modified nucleotides. In some instances, the polynucleic acidmolecule is a polynucleic acid molecule of SEQ ID NOs: 16-75, 452-1955,1956-1962, 1967-2002, 2013-2032, 2082-2109, or 2117.

In some instances, the polynucleic acid molecule comprises at least oneof: from about 5% to about 100% modification, from about 10% to about100% modification, from about 20% to about 100% modification, from about30% to about 100% modification, from about 40% to about 100%modification, from about 50% to about 100% modification, from about 60%to about 100% modification, from about 70% to about 100% modification,from about 80% to about 100% modification, and from about 90% to about100% modification. In some instances, the polynucleic acid molecule is apolynucleic acid molecule of SEQ ID NOs: 16-75, 452-1955, 1956-1962,1967-2002, 2013-2032, 2082-2109, or 2117.

In some instances, about 5 to about 100% of the polynucleic acidmolecule comprise the artificial nucleotide analogues described herein.In some instances, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of thepolynucleic acid molecule comprise the artificial nucleotide analoguesdescribed herein. In some instances, about 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%of a polynucleic acid molecule of SEQ ID NOs: 16-75, 452-1955,1956-1962, 1967-2002, 2013-2032, 2082-2109, or 2117 comprise theartificial nucleotide analogues described herein. In some instances,about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 100% of a polynucleic acid molecule ofSEQ ID NOs: 16-45 comprise the artificial nucleotide analogues describedherein. In some instances, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of apolynucleic acid molecule of SEQ ID NOs: 452-1203 comprise theartificial nucleotide analogues described herein. In some instances,about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 100% of a polynucleic acid molecule ofSEQ ID NOs: 1956-1962 comprise the artificial nucleotide analoguesdescribed herein. In some instances, about 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%of a polynucleic acid molecule of SEQ ID NOs: 1967-2002 comprise theartificial nucleotide analogues described herein. In some instances,about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 100% of a polynucleic acid molecule ofSEQ ID NOs: 2013-2032 comprise the artificial nucleotide analoguesdescribed herein. In some instances, about 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%of a polynucleic acid molecule of SEQ ID NOs: 2082-2109 or 2117 comprisethe artificial nucleotide analogues described herein. In someembodiments, the artificial nucleotide analogues include 2′-O-methyl,2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy,T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-0-DMAP),T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido(2′-O-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonatenucleotides, thiolphosphonate nucleotides, 2′-fluoroN3-P5′-phosphoramidites, or a combination thereof.

In some instances, the polynucleic acid molecule that comprises anartificial nucleotide analogue comprises SEQ ID NOs: 46-75. In someinstances, the polynucleic acid molecule that comprises an artificialnucleotide analogue comprises SEQ ID NOs: 1204-1955. In some instances,the polynucleic acid molecule that comprises an artificial nucleotideanalogue comprises SEQ ID NOs: 1967-2002. In some instances, thepolynucleic acid molecule that comprises an artificial nucleotideanalogue comprises SEQ ID NOs: 2013-2032. In some instances, thepolynucleic acid molecule that comprises an artificial nucleotideanalogue comprises SEQ ID NOs: 2082-2109 or 2117.

In some cases, one or more of the artificial nucleotide analoguesdescribed herein are resistant toward nucleases such as for exampleribonuclease such as RNase H, deoxyribunuclease such as DNase, orexonuclease such as 5′-3′ exonuclease and 3′-5′ exonuclease whencompared to natural polynucleic acid molecules. In some instances,artificial nucleotide analogues comprising 2′-O-methyl,2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy,T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido(2′-O-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonatenucleotides, thiolphosphonate nucleotides, 2′-fluoroN3-P5′-phosphoramidites, or combinations thereof are resistant towardnucleases such as for example ribonuclease such as RNase H,deoxyribunuclease such as DNase, or exonuclease such as 5′-3′exonuclease and 3′-5′ exonuclease. In some instances, 2′-O-methylmodified polynucleic acid molecule is nuclease resistant (e.g., RNase H,DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In someinstances, 2′O-methoxyethyl (2′-O-MOE) modified polynucleic acidmolecule is nuclease resistant (e.g., RNase H, DNase, 5′-3′ exonucleaseor 3′-5′ exonuclease resistant). In some instances, 2′-O-aminopropylmodified polynucleic acid molecule is nuclease resistant (e.g., RNase H,DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In someinstances, 2′-deoxy modified polynucleic acid molecule is nucleaseresistant (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonucleaseresistant). In some instances, T-deoxy-2′-fluoro modified polynucleicacid molecule is nuclease resistant (e.g., RNase H, DNase, 5′-3′exonuclease or 3′-5′ exonuclease resistant). In some instances,2′-O-aminopropyl (2′-O-AP) modified polynucleic acid molecule isnuclease resistant (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′exonuclease resistant). In some instances, 2′-O-dimethylaminoethyl(2′-O-DMAOE) modified polynucleic acid molecule is nuclease resistant(e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonucleaseresistant). In some instances, 2′-O-dimethylaminopropyl (2′-O-DMAP)modified polynucleic acid molecule is nuclease resistant (e.g., RNase H,DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In someinstances, T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE) modifiedpolynucleic acid molecule is nuclease resistant (e.g., RNase H, DNase,5′-3′ exonuclease or 3′-5′ exonuclease resistant). In some instances,2′-O—N-methylacetamido (2′-O-NMA) modified polynucleic acid molecule isnuclease resistant (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′exonuclease resistant). In some instances, LNA-modified polynucleic acidmolecule is nuclease resistant (e.g., RNase H, DNase, 5′-3′ exonucleaseor 3′-5′ exonuclease resistant). In some instances, ENA-modifiedpolynucleic acid molecule is nuclease resistant (e.g., RNase H, DNase,5′-3′ exonuclease or 3′-5′ exonuclease resistant). In some instances,HNA-modified polynucleic acid molecule is nuclease resistant (e.g.,RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant).Morpholinos may be nuclease resistant (e.g., RNase H, DNase, 5′-3′exonuclease or 3′-5′ exonuclease resistant). In some instances,PNA-modified polynucleic acid molecule is resistant to nucleases (e.g.,RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). Insome instances, methylphosphonate nucleotide-modified polynucleic acidmolecule is nuclease resistant (e.g., RNase H, DNase, 5′-3′ exonucleaseor 3′-5′ exonuclease resistant). In some instances, thiolphosphonatenucleotide-modified polynucleic acid molecule is nuclease resistant(e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonucleaseresistant). In some instances, polynucleic acid molecule comprising2′-fluoro N3-P5′-phosphoramidites is nuclease resistant (e.g., RNase H,DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistant). In someinstances, the 5′ conjugates described herein inhibit 5′-3′exonucleolytic cleavage. In some instances, the 3′ conjugates describedherein inhibit 3′-5′ exonucleolytic cleavage.

In some embodiments, one or more of the artificial nucleotide analoguesdescribed herein have increased binding affinity toward their mRNAtarget relative to an equivalent natural polynucleic acid molecule. Theone or more of the artificial nucleotide analogues comprising2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy,T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido(2′-O-NMA) modified, LNA, ENA, PNA, I-INA, morpholino, methylphosphonatenucleotides, thiolphosphonate nucleotides, or 2′-fluoroN3-P5′-phosphoramidites can have increased binding affinity toward theirmRNA target relative to an equivalent natural polynucleic acid molecule.In some instances, 2′-O-methyl modified polynucleic acid molecule hasincreased binding affinity toward their mRNA target relative to anequivalent natural polynucleic acid molecule. In some instances,2′-O-methoxyethyl (2′-O-MOE) modified polynucleic acid molecule hasincreased binding affinity toward their mRNA target relative to anequivalent natural polynucleic acid molecule. In some instances,2′-O-aminopropyl modified polynucleic acid molecule has increasedbinding affinity toward their mRNA target relative to an equivalentnatural polynucleic acid molecule. In some instances, 2′-deoxy modifiedpolynucleic acid molecule has increased binding affinity toward theirmRNA target relative to an equivalent natural polynucleic acid molecule.In some instances, T-deoxy-2′-fluoro modified polynucleic acid moleculehas increased binding affinity toward their mRNA target relative to anequivalent natural polynucleic acid molecule. In some instances,2′-O-aminopropyl (2′-O-AP) modified polynucleic acid molecule hasincreased binding affinity toward their mRNA target relative to anequivalent natural polynucleic acid molecule. In some instances,2′-O-dimethylaminoethyl (2′-O-DMAOE) modified polynucleic acid moleculehas increased binding affinity toward their mRNA target relative to anequivalent natural polynucleic acid molecule. In some instances,2′-O-dimethylaminopropyl (2′-O-DMAP) modified polynucleic acid moleculehas increased binding affinity toward their mRNA target relative to anequivalent natural polynucleic acid molecule. In some instances,T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE) modified polynucleic acidmolecule has increased binding affinity toward their mRNA targetrelative to an equivalent natural polynucleic acid molecule. In someinstances, 2′-O—N-methylacetamido (2′-O-NMA) modified polynucleic acidmolecule has increased binding affinity toward their mRNA targetrelative to an equivalent natural polynucleic acid molecule. In someinstances, LNA-modified polynucleic acid molecule has increased bindingaffinity toward their mRNA target relative to an equivalent naturalpolynucleic acid molecule. In some instances, ENA-modified polynucleicacid molecule has increased binding affinity toward their mRNA targetrelative to an equivalent natural polynucleic acid molecule. In someinstances, PNA-modified polynucleic acid molecule has increased bindingaffinity toward their mRNA target relative to an equivalent naturalpolynucleic acid molecule. In some instances, I-INA-modified polynucleicacid molecule has increased binding affinity toward their mRNA targetrelative to an equivalent natural polynucleic acid molecule. In someinstances, morpholino-modified polynucleic acid molecule has increasedbinding affinity toward their mRNA target relative to an equivalentnatural polynucleic acid molecule. In some instances, methylphosphonatenucleotide-modified polynucleic acid molecule has increased bindingaffinity toward their mRNA target relative to an equivalent naturalpolynucleic acid molecule. In some instances, thiolphosphonatenucleotide-modified polynucleic acid molecule has increased bindingaffinity toward their mRNA target relative to an equivalent naturalpolynucleic acid molecule. In some instances, polynucleic acid moleculecomprising 2′-fluoro N3-P5′-phosphoramidites has increased bindingaffinity toward their mRNA target relative to an equivalent naturalpolynucleic acid molecule. In some cases, the increased affinity isillustrated with a lower Kd, a higher melt temperature (Tm), or acombination thereof.

In some embodiments, a polynucleic acid molecule described herein is achirally pure (or stereo pure) polynucleic acid molecule, or apolynucleic acid molecule comprising a single enantiomer. In someinstances, the polynucleic acid molecule comprises L-nucleotide. In someinstances, the polynucleic acid molecule comprises D-nucleotides. Insome instance, a polynucleic acid molecule composition comprises lessthan 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of its mirrorenantiomer. In some cases, a polynucleic acid molecule compositioncomprises less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or lessof a racemic mixture. In some instances, the polynucleic acid moleculeis a polynucleic acid molecule described in: U.S. Patent PublicationNos: 2014/194610 and 2015/211006; and PCT Publication No.: WO2015107425.

In some embodiments, a polynucleic acid molecule described herein isfurther modified to include an aptamer-conjugating moiety. In someinstances, the aptamer conjugating moiety is a DNA aptamer-conjugatingmoiety. In some instances, the aptamer-conjugating moiety is Alphamer(Centauri Therapeutics), which comprises an aptamer portion thatrecognizes a specific cell-surface target and a portion that presents aspecific epitopes for attaching to circulating antibodies. In someinstance, a polynucleic acid molecule described herein is furthermodified to include an aptamer-conjugating moiety as described in: U.S.Pat. Nos. 8,604,184, 8,591,910, and 7,850,975.

In additional embodiments, a polynucleic acid molecule described hereinis modified to increase its stability. In some embodiment, thepolynucleic acid molecule is RNA (e.g., siRNA), the polynucleic acidmolecule is modified to increase its stability. In some instances, thepolynucleic acid molecule is modified by one or more of themodifications described above to increase its stability. In some cases,the polynucleic acid molecule is modified at the 2′ hydroxyl position,such as by 2′-O-methyl, 2′-O-methoxyethyl(2′-O-MOE), 2′-O-aminopropyl,2′-deoxy, T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or2′-O—N-methylacetamido (2′-O-NMA) modification or by a locked or bridgedribose conformation (e.g., LNA or ENA). In some cases, the polynucleicacid molecule is modified by 2′-O-methyl and/or 2′-O-methoxyethylribose. In some cases, the polynucleic acid molecule also includesmorpholinos, PNAs, HNA, methylphosphonate nucleotides, thiolphosphonatenucleotides, and/or 2′-fluoro N3-P5′-phosphoramidites to increase itsstability. In some instances, the polynucleic acid molecule is achirally pure (or stereo pure) polynucleic acid molecule. In someinstances, the chirally pure (or stereo pure) polynucleic acid moleculeis modified to increase its stability. Suitable modifications to the RNAto increase stability for delivery will be apparent to the skilledperson.

In some embodiments, a polynucleic acid molecule described herein hasRNAi activity that modulates expression of RNA encoded by a genedescribed supra. In some instances, a polynucleic acid moleculedescribed herein is a double-stranded siRNA molecule that down-regulatesexpression of a gene, wherein one of the strands of the double-strandedsiRNA molecule comprises a nucleotide sequence that is complementary toa nucleotide sequence of the gene or RNA encoded by the gene or aportion thereof, and wherein the second strand of the double-strandedsiRNA molecule comprises a nucleotide sequence substantially similar tothe nucleotide sequence of the gene or RNA encoded by the gene or aportion thereof. In some cases, a polynucleic acid molecule describedherein is a double-stranded siRNA molecule that down-regulatesexpression of a gene, wherein each strand of the siRNA moleculecomprises about 15 to 25, 18 to 24, or 19 to about 23 nucleotides, andwherein each strand comprises at least about 14, 17, or 19 nucleotidesthat are complementary to the nucleotides of the other strand. In somecases, a polynucleic acid molecule described herein is a double-strandedsiRNA molecule that down-regulates expression of a gene, wherein eachstrand of the siRNA molecule comprises about 19 to about 23 nucleotides,and wherein each strand comprises at least about 19 nucleotides that arecomplementary to the nucleotides of the other strand. In some instances,the gene is KRAS, EGFR, AR, HPRT1, CNNTB1 (β-catenin), or β-cateninassociated genes.

In some embodiments, a polynucleic acid molecule described herein isconstructed using chemical synthesis and/or enzymatic ligation reactionsusing procedures known in the art. For example, a polynucleic acidmolecule is chemically synthesized using naturally occurring nucleotidesor variously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed between the polynucleic acid molecule and target nucleicacids. Exemplary methods include those described in: U.S. Pat. Nos.5,142,047; 5,185,444; 5,889,136; 6,008,400; and 6,111,086; PCTPublication No. WO2009099942; or European Publication No. 1579015.Additional exemplary methods include those described in: Griffey et al.,“2′-O-aminopropyl ribonucleotides: a zwitterionic modification thatenhances the exonuclease resistance and biological activity of antisenseoligonucleotides,” J. Med. Chem. 39(26):5100-5109 (1997)); Obika, et al.“Synthesis of 2′-0,4′-C-methyleneuridine and -cytidine. Novel bicyclicnucleosides having a fixed C3, -endo sugar puckering”. TetrahedronLetters 38 (50): 8735 (1997); Koizumi, M. “ENA oligonucleotides astherapeutics”. Current opinion in molecular therapeutics 8 (2): 144-149(2006); and Abramova et al., “Novel oligonucleotide analogues based onmorpholino nucleoside subunits-antisense technologies: new chemicalpossibilities,” Indian Journal of Chemistry 48B:1721-1726 (2009).Alternatively, the polynucleic acid molecule is produced biologicallyusing an expression vector into which a polynucleic acid molecule hasbeen subcloned in an antisense orientation (i.e., RNA transcribed fromthe inserted polynucleic acid molecule will be of an antisenseorientation to a target polynucleic acid molecule of interest).

Conjugation Chemistry

In some embodiments, a polynucleic acid molecule is conjugated to abinding moiety. In some instances, the binding moiety comprises aminoacids, peptides, polypeptides, proteins, antibodies, antigens, toxins,hormones, lipids, nucleotides, nucleosides, sugars, carbohydrates,polymers such as polyethylene glycol and polypropylene glycol, as wellas analogs or derivatives of all of these classes of substances.Additional examples of binding moiety also include steroids, such ascholesterol, phospholipids, di- and triacylglycerols, fatty acids,hydrocarbons (e.g., saturated, unsaturated, or contains substitutions),enzyme substrates, biotin, digoxigenin, and polysaccharides. In someinstances, the binding moiety is an antibody or binding fragmentthereof. In some instances, the polynucleic acid molecule is furtherconjugated to a polymer, and optionally an endosomolytic moiety.

In some embodiments, the polynucleic acid molecule is conjugated to thebinding moiety by a chemical ligation process. In some instances, thepolynucleic acid molecule is conjugated to the binding moiety by anative ligation. In some instances, the conjugation is as described in:Dawson, et al. “Synthesis of proteins by native chemical ligation,”Science 1994, 266, 776-779; Dawson, et al. “Modulation of Reactivity inNative Chemical Ligation through the Use of Thiol Additives,” J. Am.Chem. Soc. 1997, 119, 4325-4329; Hackeng, et al. “Protein synthesis bynative chemical ligation: Expanded scope by using straightforwardmethodology,” Proc. Natl. Acad. Sci. USA 1999, 96, 10068-10073; or Wu,et al. “Building complex glycopeptides: Development of a cysteine-freenative chemical ligation protocol,” Angew. Chem. Int. Ed. 2006, 45,4116-4125. In some instances, the conjugation is as described in U.S.Pat. No. 8,936,910. In some embodiments, the polynucleic acid moleculeis conjugated to the binding moiety either site-specifically ornon-specifically via native ligation chemistry.

In some instances, the polynucleic acid molecule is conjugated to thebinding moiety by a site-directed method utilizing a “traceless”coupling technology (Philochem). In some instances, the “traceless”coupling technology utilizes an N-terminal 1,2-aminothiol group on thebinding moiety which is then conjugate with a polynucleic acid moleculecontaining an aldehyde group. (see Casi et al., “Site-specific tracelesscoupling of potent cytotoxic drugs to recombinant antibodies forpharmacodelivery,” JACS 134(13): 5887-5892 (2012))

In some instances, the polynucleic acid molecule is conjugated to thebinding moiety by a site-directed method utilizing an unnatural aminoacid incorporated into the binding moiety. In some instances, theunnatural amino acid comprises p-acetylphenylalanine (pAcPhe). In someinstances, the keto group of pAcPhe is selectively coupled to analkoxy-amine derivatived conjugating moiety to form an oxime bond. (seeAxup et al., “Synthesis of site-specific antibody-drug conjugates usingunnatural amino acids,” PNAS 109(40): 16101-16106 (2012)).

In some instances, the polynucleic acid molecule is conjugated to thebinding moiety by a site-directed method utilizing an enzyme-catalyzedprocess. In some instances, the site-directed method utilizes SMARTag™technology (Redwood). In some instances, the SMARTag™ technologycomprises generation of a formylglycine (FGly) residue from cysteine byformylglycine-generating enzyme (FGE) through an oxidation process underthe presence of an aldehyde tag and the subsequent conjugation of FGlyto an alkylhydraine-functionalized polynucleic acid molecule viahydrazino-Pictet-Spengler (HIPS) ligation. (see Wu et al.,“Site-specific chemical modification of recombinant proteins produced inmammalian cells by using the genetically encoded aldehyde tag,” PNAS106(9): 3000-3005 (2009); Agarwal, et al., “A Pictet-Spengler ligationfor protein chemical modification,” PNAS 110(1): 46-51 (2013))

In some instances, the enzyme-catalyzed process comprises microbialtransglutaminase (mTG). In some cases, the polynucleic acid molecule isconjugated to the binding moiety utilizing a microbial transglutaminzecatalyzed process. In some instances, mTG catalyzes the formation of acovalent bond between the amide side chain of a glutamine within therecognition sequence and a primary amine of a functionalized polynucleicacid molecule. In some instances, mTG is produced from Streptomycesmobarensis. (see Strop et al., “Location matters: site of conjugationmodulates stability and pharmacokinetics of antibody drug conjugates,”Chemistry and Biology 20(2) 161-167 (2013))

In some instances, the polynucleic acid molecule is conjugated to thebinding moiety by a method as described in PCT Publication No.WO2014/140317, which utilizes a sequence-specific transpeptidase.

In some instances, the polynucleic acid molecule is conjugated to thebinding moiety by a method as described in U.S. Patent Publication Nos.2015/0105539 and 2015/0105540.

Binding Moiety

In some embodiments, the binding moiety A is a polypeptide. In someinstances, the polypeptide is an antibody or its fragment thereof. Insome cases, the fragment is a binding fragment. In some instances, theantibody or binding fragment thereof comprises a humanized antibody orbinding fragment thereof, murine antibody or binding fragment thereof,chimeric antibody or binding fragment thereof, monoclonal antibody orbinding fragment thereof, monovalent Fab′, divalent Fab₂, F(ab)′₃fragments, single-chain variable fragment (scFv), bis-scFv, (scFv)₂,diabody, minibody, nanobody, triabody, tetrabody, disulfide stabilizedFv protein (dsFv), single-domain antibody (sdAb), Ig NAR, camelidantibody or binding fragment thereof, bispecific antibody or bidingfragment thereof, or a chemically modified derivative thereof.

In some instances, A is an antibody or binding fragment thereof. In someinstances, A is a humanized antibody or binding fragment thereof, murineantibody or binding fragment thereof, chimeric antibody or bindingfragment thereof, monoclonal antibody or binding fragment thereof,monovalent Fab′, divalent Fab₂, F(ab)′₃ fragments, single-chain variablefragment (scFv), bis-scFv, (scFv)₂, diabody, minibody, nanobody,triabody, tetrabody, disulfide stabilized Fv protein (“dsFv”),single-domain antibody (sdAb), Ig NAR, camelid antibody or bindingfragment thereof, bispecific antibody or biding fragment thereof, or achemically modified derivative thereof. In some instances, A is ahumanized antibody or binding fragment thereof. In some instances, A isa murine antibody or binding fragment thereof. In some instances, A is achimeric antibody or binding fragment thereof. In some instances, A is amonoclonal antibody or binding fragment thereof. In some instances, A isa monovalent Fab′. In some instances, A is a diavalent Fab₂. In someinstances, A is a single-chain variable fragment (scFv).

In some embodiments, the binding moiety A is a bispecific antibody orbinding fragment thereof. In some instances, the bispecific antibody isa trifunctional antibody or a bispecific mini-antibody. In some cases,the bispecific antibody is a trifunctional antibody. In some instances,the trifunctional antibody is a full length monoclonal antibodycomprising binding sites for two different antigens. Exemplarytrifunctional antibodies include catumaxomab (which targets EpCAM andCD3; Fresenius Biotech/Trion Pharma), ertumaxomab (targets HER2/neu/CD3;Fresenius Biotech/Trion Pharma), lymphomun FBTA05 (targets CD20/CD3;Fresenius Biotech/Trion Pharma), RG7221 (R05520985; targets Angiopoietin2/VEGF; Roche), RG7597 (targets Her1/Her3; Genentech/Roche), MM141(targets IGF1R/Her3; Merrimack), ABT122 (targets TNFα/IL17; Abbvie),ABT981 (targets IL1α/IL1β; Abbott), LY3164530 (targets Her1/cMET; EliLilly), and TRBS07 (Ektomab; targets GD2/CD3; Trion Research Gmbh).Additional exemplary trifunctional antibodies include mAb² from F-starBiotechnology Ltd. In some instances, A is a bispecific trifunctionalantibody. In some embodiments, A is a bispecific trifunctional antibodyselected from: catumaxomab (which targets EpCAM and CD3; FreseniusBiotech/Trion Pharma), ertumaxomab (targets HER2/neu/CD3; FreseniusBiotech/Trion Pharma), lymphomun FBTA05 (targets CD20/CD3; FreseniusBiotech/Trion Pharma), RG7221 (R05520985; targets Angiopoietin 2/VEGF;Roche), RG7597 (targets Her1/Her3; Genentech/Roche), MM141 (targetsIGF1R/Her3; Merrimack), ABT122 (targets TNFα/IL17; Abbvie), ABT981(targets IL1α/IL1β; Abbott), LY3164530 (targets Her1/cMET; Eli Lilly),TRBS07 (Ektomab; targets GD2/CD3; Trion Research Gmbh), and a mAb² fromF-star Biotechnology Ltd.

In some cases, the bispecific antibody is a bispecific mini-antibody. Insome instances, the bispecific mini-antibody comprises divalent Fab₂,F(ab)′₃ fragments, bis-scFv, (scFv)₂, diabody, minibody, triabody,tetrabody or a bi-specific T-cell engager (BiTE). In some embodiments,the bi-specific T-cell engager is a fusion protein that contains twosingle-chain variable fragments (scFvs) in which the two scFvs targetepitopes of two different antigens. Exemplary bispecific mini-antibodiesinclude, but are not limited to, DART (dual-affinity re-targetingplatform; MacroGenics), blinatumomab (MT103 or AMG103; which targetsCD19/CD3; Micromet), MT111 (targets CEA/CD3; Micromet/Amegen), MT112(BAY2010112; targets PSMA/CD3; Micromet/Bayer), MT110 (AMG 110; targetsEPCAM/CD3; Amgen/Micromet), MGD006 (targets CD123/CD3; MacroGenics),MGD007 (targets GPA33/CD3; MacroGenics), BI1034020 (targets twodifferent epitopes on β-amyloid; Ablynx), ALX0761 (targets IL17A/IL17F;Ablynx), TF2 (targets CEA/hepten; Immunomedics), IL-17/IL-34 biAb (BMS),AFM13 (targets CD30/CD16; Affimed), AFM11 (targets CD19/CD3; Affimed),and domain antibodies (dAbs from Domantis/GSK).

In some embodiments, the binding moiety A is a bispecific mini-antibody.In some instances, A is a bispecific Fab₂. In some instances, A is abispecific F(ab)′₃ fragment. In some cases, A is a bispecific bis-scFv.In some cases, A is a bispecific (scFv)₂. In some embodiments, A is abispecific diabody. In some embodiments, A is a bispecific minibody. Insome embodiments, A is a bispecific triabody. In other embodiments, A isa bispecific tetrabody. In other embodiments, A is a bi-specific T-cellengager (BiTE). In additional embodiments, A is a bispecificmini-antibody selected from: DART (dual-affinity re-targeting platform;MacroGenics), blinatumomab (MT103 or AMG103; which targets CD19/CD3;Micromet), MT111 (targets CEA/CD3; Micromet/Amegen), MT112 (BAY2010112;targets PSMA/CD3; Micromet/Bayer), MT110 (AMG 110; targets EPCAM/CD3;Amgen/Micromet), MGD006 (targets CD123/CD3; MacroGenics), MGD007(targets GPA33/CD3; MacroGenics), BI1034020 (targets two differentepitopes on β-amyloid; Ablynx), ALX0761 (targets IL17A/IL17F; Ablynx),TF2 (targets CEA/hepten; Immunomedics), IL-17/IL-34 biAb (BMS), AFM13(targets CD30/CD16; Affimed), AFM11 (targets CD19/CD3; Affimed), anddomain antibodies (dAbs from Domantis/GSK).

In some embodiments, the binding moiety A is a trispecific antibody. Insome instances, the trispecific antibody comprises F(ab)′₃ fragments ora triabody. In some instances, A is a trispecific F(ab)′₃ fragment. Insome cases, A is a triabody. In some embodiments, A is a trispecificantibody as described in Dimas, et al., “Development of a trispecificantibody designed to simultaneously and efficiently target threedifferent antigens on tumor cells,” Mol. Pharmaceutics, 12(9): 3490-3501(2015).

In some embodiments, the binding moiety A is an antibody or bindingfragment thereof that recognizes a cell surface protein. In someinstances, the cell surface protein is an antigen expressed by acancerous cell. Exemplary cancer antigens include, but are not limitedto, alpha fetoprotein, ASLG659, B7-H3, BAFF-R, Brevican, CA125 (MUC16),CA15-3, CA19-9, carcinoembryonic antigen (CEA), CA242, CRIPTO (CR, CR1,CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor), CTLA-4,CXCR5, E16 (LAT1, SLC7A5), FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domaincontaining phosphatase anchor protein 1a), SPAP1B, SPAP1C), epidermalgrowth factor, ETBR, Fc receptor-like protein 1 (FCRH1), GEDA, HLA-DOB(Beta subunit of MHC class II molecule (Ia antigen), human chorionicgonadotropin, ICOS, IL-2 receptor, IL20Ra, Immunoglobulin superfamilyreceptor translocation associated 2 (IRTA2), L6, Lewis Y, Lewis X,MAGE-1, MAGE-2, MAGE-3, MAGE 4, MARTI, mesothelin, MDP, MPF (SMR, MSLN),MCP1 (CCL2), macrophage inhibitory factor (MIF), MPG, MSG783, mucin,MUC1-KLH, Napi3b (SLC34A2), nectin-4, Neu oncogene product, NCA,placental alkaline phosphatase, prostate specific membrane antigen(PMSA), prostatic acid phosphatase, PSCA hlg, p97, Purinergic receptorP2X ligand-gated ion channel 5 (P2X5), LY64 (Lymphocyte antigen 64(RP105), gp100, P21, six transmembrane epithelial antigen of prostate(STEAP1), STEAP2, Sema 5b, tumor-associated glycoprotein 72 (TAG-72),TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potentialcation channel, subfamily M, member 4) and the like.

In some instances, the cell surface protein comprises clusters ofdifferentiation (CD) cell surface markers. Exemplary CD cell surfacemarkers include, but are not limited to, CD1, CD2, CD3, CD4, CD5, CD6,CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CDw12, CD13, CD14,CD15, CD15s, CD16, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24,CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36,CD37, CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD45RO, CD45RA,CD45RB, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f,CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CDw60, CD61,CD62E, CD62L (L-selectin), CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c,CD66d, CD66e, CD79 (e.g., CD79a, CD79b), CD90, CD95 (Fas), CD103, CD104,CD125 (IL5RA), CD134 (OX40), CD137 (4-1BB), CD152 (CTLA-4), CD221,CD274, CD279 (PD-1), CD319 (SLAMF7), CD326 (EpCAM), and the like.

In some instances, the binding moiety A is an antibody or bindingfragment thereof that recognizes a cancer antigen. In some instances,the binding moiety A is an antibody or binding fragment thereof thatrecognizes alpha fetoprotein, ASLG659, B7-H3, BAFF-R, Brevican, CA125(MUC16), CA15-3, CA19-9, carcinoembryonic antigen (CEA), CA242, CRIPTO(CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor),CTLA-4, CXCR5, E16 (LAT1, SLC7A5), FcRH2 (IFGP4, IRTA4, SPAP1A (SH2domain containing phosphatase anchor protein 1a), SPAP1B, SPAP1C),epidermal growth factor, ETBR, Fc receptor-like protein 1 (FCRH1), GEDA,HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen), humanchorionic gonadotropin, ICOS, IL-2 receptor, IL20Ra, Immunoglobulinsuperfamily receptor translocation associated 2 (IRTA2), L6, Lewis Y,Lewis X, MAGE-1, MAGE-2, MAGE-3, MAGE 4, MARTI, mesothelin, MCP1 (CCL2),MDP, macrophage inhibitory factor (MIF), MPF (SMR, MSLN), MPG, MSG783,mucin, MUC1-KLH, Napi3b (SLC34A2), nectin-4, Neu oncogene product, NCA,placental alkaline phosphatase, prostate specific membrane antigen(PMSA), prostatic acid phosphatase, PSCA hlg, p97, Purinergic receptorP2X ligand-gated ion channel 5 (P2X5), LY64 (Lymphocyte antigen 64(RP105), gp100, P21, six transmembrane epithelial antigen of prostate(STEAP1), STEAP2, Sema 5b, tumor-associated glycoprotein 72 (TAG-72),TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potentialcation channel, subfamily M, member 4) or a combination thereof.

In some instances, the binding moiety A is an antibody or bindingfragment thereof that recognizes a CD cell surface marker. In someinstances, the binding moiety A is an antibody or binding fragmentthereof that recognizes CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9,CD10, CD11a, CD11b, CD11c, CD11d, CDw12, CD13, CD14, CD15, CD15s, CD16,CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28,CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40,CD41, CD42, CD43, CD44, CD45, CD45RO, CD45RA, CD45RB, CD46, CD47, CD48,CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54,CD55, CD56, CD57, CD58, CD59, CDw60, CD61, CD62E, CD62L (L-selectin),CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66d, CD66e, CD79 (e.g.,CD79a, CD79b), CD90, CD95 (Fas), CD103, CD104, CD125 (IL5RA), CD134(OX40), CD137 (4-1BB), CD152 (CTLA-4), CD221, CD274, CD279 (PD-1), CD319(SLAMF7), CD326 (EpCAM), or a combination thereof.

In some embodiments, the antibody or binding fragment thereof compriseszalutumumab (HuMax-EFGr, Genmab), abagovomab (Menarini), abituzumab(Merck), adecatumumab (MT201), alacizumab pegol, alemtuzumab (Campath®,MabCampath, or Campath-1H; Leukosite), AlloMune (BioTransplant),amatuximab (Morphotek, Inc.), anti-VEGF (Genetech), anatumomabmafenatox, apolizumab (hu1D10), ascrinvacumab (Pfizer Inc.),atezolizumab (MPDL3280A; Genentech/Roche), B43.13 (OvaRex, AltaRexCorporation), basiliximab (Simulect®, Novartis), belimumab (Benlysta®,GlaxoSmithKline), bevacizumab (Avastin®, Genentech), blinatumomab(Blincyto, AMG103; Amgen), BEC2 (ImGlone Systems Inc.), carlumab(Janssen Biotech), catumaxomab (Removab, Trion Pharma), CEAcide(Immunomedics), Cetuximab (Erbitux®, ImClone), citatuzumab bogatox(VB6-845), cixutumumab (IMC-A12, ImClone Systems Inc.), conatumumab (AMG655, Amgen), dacetuzumab (SGN-40, huS2C6; Seattle Genetics, Inc.),daratumumab (Darzalex®, Janssen Biotech), detumomab, drozitumab(Genentech), durvalumab (MedImmune), dusigitumab (MedImmune),edrecolomab (MAb17-1A, Panorex, Glaxo Wellcome), elotuzumab (Empliciti™,Bristol-Myers Squibb), emibetuzumab (Eli Lilly), enavatuzumab (FacetBiotech Corp.), enfortumab vedotin (Seattle Genetics, Inc.),enoblituzumab (MGA271, MacroGenics, Inc.), ensituxumab (NeogenixOncology, Inc.), epratuzumab (LymphoCide, Immunomedics, Inc.),ertumaxomab (Rexomun®, Trion Pharma), etaracizumab (Abegrin, MedImmune),farletuzumab (MORAb-003, Morphotek, Inc), FBTA05 (Lymphomun, TrionPharma), ficlatuzumab (AVEO Pharmaceuticals), figitumumab (CP-751871,Pfizer), flanvotumab (ImClone Systems), fresolimumab (GC1008,Aanofi-Aventis), futuximab, glaximab, ganitumab (Amgen), girentuximab(Rencarex®, Wilex AG), IMAB362 (Claudiximab, Ganymed PharmaceuticalsAG), imalumab (Baxalta), IMC-1C11 (ImClone Systems), IMC-C225 (ImcloneSystems Inc.), imgatuzumab (Genentech/Roche), intetumumab (Centocor,Inc.), ipilimumab (Yervoy®, Bristol-Myers Squibb), iratumumab (Medarex,Inc.), isatuximab (SAR650984, Sanofi-Aventis), labetuzumab (CEA-CIDE,Immunomedics), lexatumumab (ETR2-ST01, Cambridge Antibody Technology),lintuzumab (SGN-33, Seattle Genetics), lucatumumab (Novartis),lumiliximab, mapatumumab (HGS-ETR1, Human Genome Sciences), matuzumab(EMD 72000, Merck), milatuzumab (hLL1, Immunomedics, Inc.), mitumomab(BEC-2, ImClone Systems), narnatumab (ImClone Systems), necitumumab(Portrazza™, Eli Lilly), nesvacumab (Regeneron Pharmaceuticals),nimotuzumab (h-R3, BIOMAb EGFR, TheraClM, Theraloc, or CIMAher; BiotechPharmaceutical Co.), nivolumab (Opdivo®, Bristol-Myers Squibb),obinutuzumab (Gazyva or Gazyvaro; Hoffmann-La Roche), ocaratuzumab(AME-133v, LY2469298; Mentrik Biotech, LLC), ofatumumab (Arzerra®,Genmab), onartuzumab (Genentech), Ontuxizumab (Morphotek, Inc.),oregovomab (OvaRex®, AltaRex Corp.), otlertuzumab (EmergentBioSolutions), panitumumab (ABX-EGF, Amgen), pankomab (Glycotope GMBH),parsatuzumab (Genentech), patritumab, pembrolizumab (Keytruda®, Merck),pemtumomab (Theragyn, Antisoma), pertuzumab (Perjeta, Genentech),pidilizumab (CT-011, Medivation), polatuzumab vedotin (Genentech/Roche),pritumumab, racotumomab (Vaxira®, Recombio), ramucirumab (Cyramza®,ImClone Systems Inc.), rituximab (Rituxan®, Genentech), robatumumab(Schering-Plough), Seribantumab (Sanofi/Merrimack Pharmaceuticals,Inc.), sibrotuzumab, siltuximab (Sylvant™, Janssen Biotech), Smart MI95(Protein Design Labs, Inc.), Smart ID10 (Protein Design Labs, Inc.),tabalumab (LY2127399, Eli Lilly), taplitumomab paptox, tenatumomab,teprotumumab (Roche), tetulomab, TGN1412 (CD28-SuperMAB or TAB08),tigatuzumab (CD-1008, Daiichi Sankyo), tositumomab, trastuzumab(Herceptin®), tremelimumab (CP-672,206; Pfizer), tucotuzumab celmoleukin(EMD Pharmaceuticals), ublituximab, urelumab (BMS-663513, Bristol-MyersSquibb), volociximab (M200, Biogen Idec), zatuximab, and the like.

In some embodiments, the binding moiety A comprises zalutumumab(HuMax-EFGr, Genmab), abagovomab (Menarini), abituzumab (Merck),adecatumumab (MT201), alacizumab pegol, alemtuzumab (Campath®,MabCampath, or Campath-1H; Leukosite), AlloMune (BioTransplant),amatuximab (Morphotek, Inc.), anti-VEGF (Genetech), anatumomabmafenatox, apolizumab (hu1D10), ascrinvacumab (Pfizer Inc.),atezolizumab (MPDL3280A; Genentech/Roche), B43.13 (OvaRex, AltaRexCorporation), basiliximab (Simulect®, Novartis), belimumab (Benlysta®,GlaxoSmithKline), bevacizumab (Avastin®, Genentech), blinatumomab(Blincyto, AMG103; Amgen), BEC2 (ImGlone Systems Inc.), carlumab(Janssen Biotech), catumaxomab (Removab, Trion Pharma), CEAcide(Immunomedics), Cetuximab (Erbitux®, ImClone), citatuzumab bogatox(VB6-845), cixutumumab (IMC-A12, ImClone Systems Inc.), conatumumab (AMG655, Amgen), dacetuzumab (SGN-40, huS2C6; Seattle Genetics, Inc.),daratumumab (Darzalex®, Janssen Biotech), detumomab, drozitumab(Genentech), durvalumab (MedImmune), dusigitumab (MedImmune),edrecolomab (MAb17-1A, Panorex, Glaxo Wellcome), elotuzumab (Empliciti™,Bristol-Myers Squibb), emibetuzumab (Eli Lilly), enavatuzumab (FacetBiotech Corp.), enfortumab vedotin (Seattle Genetics, Inc.),enoblituzumab (MGA271, MacroGenics, Inc.), ensituxumab (NeogenixOncology, Inc.), epratuzumab (LymphoCide, Immunomedics, Inc.),ertumaxomab (Rexomun®, Trion Pharma), etaracizumab (Abegrin, MedImmune),farletuzumab (MORAb-003, Morphotek, Inc), FBTA05 (Lymphomun, TrionPharma), ficlatuzumab (AVEO Pharmaceuticals), figitumumab (CP-751871,Pfizer), flanvotumab (ImClone Systems), fresolimumab (GC1008,Aanofi-Aventis), futuximab, glaximab, ganitumab (Amgen), girentuximab(Rencarex®, Wilex AG), IMAB362 (Claudiximab, Ganymed PharmaceuticalsAG), imalumab (Baxalta), IMC-1C11 (ImClone Systems), IMC-C225 (ImcloneSystems Inc.), imgatuzumab (Genentech/Roche), intetumumab (Centocor,Inc.), ipilimumab (Yervoy®, Bristol-Myers Squibb), iratumumab (Medarex,Inc.), isatuximab (SAR650984, Sanofi-Aventis), labetuzumab (CEA-CIDE,Immunomedics), lexatumumab (ETR2-ST01, Cambridge Antibody Technology),lintuzumab (SGN-33, Seattle Genetics), lucatumumab (Novartis),lumiliximab, mapatumumab (HGS-ETR1, Human Genome Sciences), matuzumab(EMD 72000, Merck), milatuzumab (hLL1, Immunomedics, Inc.), mitumomab(BEC-2, ImClone Systems), narnatumab (ImClone Systems), necitumumab(Portrazza™, Eli Lilly), nesvacumab (Regeneron Pharmaceuticals),nimotuzumab (h-R3, BIOMAb EGFR, TheraClM, Theraloc, or CIMAher; BiotechPharmaceutical Co.), nivolumab (Opdivo®, Bristol-Myers Squibb),obinutuzumab (Gazyva or Gazyvaro; Hoffmann-La Roche), ocaratuzumab(AME-133v, LY2469298; Mentrik Biotech, LLC), ofatumumab (Arzerra®,Genmab), onartuzumab (Genentech), Ontuxizumab (Morphotek, Inc.),oregovomab (OvaRex®, AltaRex Corp.), otlertuzumab (EmergentBioSolutions), panitumumab (ABX-EGF, Amgen), pankomab (Glycotope GMBH),parsatuzumab (Genentech), patritumab, pembrolizumab (Keytruda®, Merck),pemtumomab (Theragyn, Antisoma), pertuzumab (Perjeta, Genentech),pidilizumab (CT-011, Medivation), polatuzumab vedotin (Genentech/Roche),pritumumab, racotumomab (Vaxira®, Recombio), ramucirumab (Cyramza®,ImClone Systems Inc.), rituximab (Rituxan®, Genentech), robatumumab(Schering-Plough), Seribantumab (Sanofi/Merrimack Pharmaceuticals,Inc.), sibrotuzumab, siltuximab (Sylvant™, Janssen Biotech), Smart MI95(Protein Design Labs, Inc.), Smart ID10 (Protein Design Labs, Inc.),tabalumab (LY2127399, Eli Lilly), taplitumomab paptox, tenatumomab,teprotumumab (Roche), tetulomab, TGN1412 (CD28-SuperMAB or TAB08),tigatuzumab (CD-1008, Daiichi Sankyo), tositumomab, trastuzumab(Herceptin®), tremelimumab (CP-672,206; Pfizer), tucotuzumab celmoleukin(EMD Pharmaceuticals), ublituximab, urelumab (BMS-663513, Bristol-MyersSquibb), volociximab (M200, Biogen Idec), or zatuximab. In someembodiments, the binding moiety A is zalutumumab (HuMax-EFGr, byGenmab).

In some embodiments, the binding moiety A is conjugated according toFormula (I) to a polynucleic acid molecule (B), and a polymer (C), andoptionally an endosomolytic moiety (D) according to Formula (II)described herein. In some instances, the polynucleic acid moleculecomprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to asequence listed in Tables 2, 3, 5, 6, 7, 9, or 11. In some embodiments,the polynucleic acid molecule comprises a sequence having at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to SEQ ID NOs: 16-75, 452-1955, 1956-1962, 1967-2002,2013-2032, 2082-2109, or 2117. In some instances, the polynucleic acidmolecule comprises a sequence selected from SEQ ID NOs: 16-75, 452-1955,1956-1962, 1967-2002, 2013-2032, 2082-2109, or 2117. In some instances,the polymer C comprises polyalkylen oxide (e.g., polyethylene glycol).In some embodiments, the endosomolytic moiety D comprises INF7 ormelittin, or their respective derivatives.

In some embodiments, the binding moiety A is conjugated to a polynucleicacid molecule (B), and a polymer (C), and optionally an endosomolyticmoiety (D) as illustrated in FIGS. 1A-1V. In some instances, the bindingmoiety A is an antibody or binding fragment thereof.

In some embodiments, the binding moiety A is conjugated to a polynucleicacid molecule (B) non-specifically. In some instances, the bindingmoiety A is conjugated to a polynucleic acid molecule (B) via a lysineresidue or a cysteine residue, in a non-site specific manner. In someinstances, the binding moiety A is conjugated to a polynucleic acidmolecule (B) via a lysine residue in a non-site specific manner. In somecases, the binding moiety A is conjugated to a polynucleic acid molecule(B) via a cysteine residue in a non-site specific manner. In someinstances, the binding moiety A is an antibody or binding fragmentthereof.

In some embodiments, the binding moiety A is conjugated to a polynucleicacid molecule (B) in a site-specific manner. In some instances, thebinding moiety A is conjugated to a polynucleic acid molecule (B)through a lysine residue, a cysteine residue, at the 5′-terminus, at the3′-terminus, an unnatural amino acid, or an enzyme-modified orenzyme-catalyzed residue, via a site-specific manner. In some instances,the binding moiety A is conjugated to a polynucleic acid molecule (B)through a lysine residue via a site-specific manner. In some instances,the binding moiety A is conjugated to a polynucleic acid molecule (B)through a cysteine residue via a site-specific manner. In someinstances, the binding moiety A is conjugated to a polynucleic acidmolecule (B) at the 5′-terminus via a site-specific manner. In someinstances, the binding moiety A is conjugated to a polynucleic acidmolecule (B) at the 3′-terminus via a site-specific manner. In someinstances, the binding moiety A is conjugated to a polynucleic acidmolecule (B) through an unnatural amino acid via a site-specific manner.In some instances, the binding moiety A is conjugated to a polynucleicacid molecule (B) through an enzyme-modified or enzyme-catalyzed residuevia a site-specific manner. In some instances, the binding moiety A isan antibody or binding fragment thereof.

In some embodiments, one or more regions of a binding moiety A (e.g., anantibody or binding fragment thereof) is conjugated to a polynucleicacid molecule (B). In some instances, the one or more regions of abinding moiety A comprise the N-terminus, the C-terminus, in theconstant region, at the hinge region, or the Fc region of the bindingmoiety A. In some instances, the polynucleic acid molecule (B) isconjugated to the N-terminus of the binding moiety A (e.g., theN-terminus of an antibody or binding fragment thereof). In someinstances, the polynucleic acid molecule (B) is conjugated to theC-terminus of the binding moiety A (e.g., the N-terminus of an antibodyor binding fragment thereof). In some instances, the polynucleic acidmolecule (B) is conjugated to the constant region of the binding moietyA (e.g., the constant region of an antibody or binding fragmentthereof). In some instances, the polynucleic acid molecule (B) isconjugated to the hinge region of the binding moiety A (e.g., theconstant region of an antibody or binding fragment thereof). In someinstances, the polynucleic acid molecule (B) is conjugated to the Fcregion of the binding moiety A (e.g., the constant region of an antibodyor binding fragment thereof).

In some embodiments, one or more polynucleic acid molecule (B) isconjugated to a binding moiety A. In some instances, about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more polynucleic acidmolecules are conjugated to one binding moiety A. In some instances,about 1 polynucleic acid molecule is conjugated to one binding moiety A.In some instances, about 2 polynucleic acid molecules are conjugated toone binding moiety A. In some instances, about 3 polynucleic acidmolecules are conjugated to one binding moiety A. In some instances,about 4 polynucleic acid molecules are conjugated to one binding moietyA. In some instances, about 5 polynucleic acid molecules are conjugatedto one binding moiety A. In some instances, about 6 polynucleic acidmolecules are conjugated to one binding moiety A. In some instances,about 7 polynucleic acid molecules are conjugated to one binding moietyA. In some instances, about 8 polynucleic acid molecules are conjugatedto one binding moiety A. In some instances, about 9 polynucleic acidmolecules are conjugated to one binding moiety A. In some instances,about 10 polynucleic acid molecules are conjugated to one binding moietyA. In some instances, about 11 polynucleic acid molecules are conjugatedto one binding moiety A. In some instances, about 12 polynucleic acidmolecules are conjugated to one binding moiety A. In some instances,about 13 polynucleic acid molecules are conjugated to one binding moietyA. In some instances, about 14 polynucleic acid molecules are conjugatedto one binding moiety A. In some instances, about 15 polynucleic acidmolecules are conjugated to one binding moiety A. In some instances,about 16 polynucleic acid molecules are conjugated to one binding moietyA. In some cases, the one or more polynucleic acid molecules are thesame. In other cases, the one or more polynucleic acid molecules aredifferent. In some instances, the binding moiety A is an antibody orbinding fragment thereof.

In some embodiments, the number of polynucleic acid molecule (B)conjugated to a binding moiety A (e.g., an antibody or binding fragmentthereof) forms a ratio. In some instances, the ratio is referred to as aDAR (drug-to-antibody) ratio, in which the drug as referred to herein isthe polynucleic acid molecule (B). In some instances, the DAR ratio ofthe polynucleic acid molecule (B) to binding moiety A is about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or greater. In someinstances, the DAR ratio of the polynucleic acid molecule (B) to bindingmoiety A is about 1 or greater. In some instances, the DAR ratio of thepolynucleic acid molecule (B) to binding moiety A is about 2 or greater.In some instances, the DAR ratio of the polynucleic acid molecule (B) tobinding moiety A is about 3 or greater. In some instances, the DAR ratioof the polynucleic acid molecule (B) to binding moiety A is about 4 orgreater. In some instances, the DAR ratio of the polynucleic acidmolecule (B) to binding moiety A is about 5 or greater. In someinstances, the DAR ratio of the polynucleic acid molecule (B) to bindingmoiety A is about 6 or greater. In some instances, the DAR ratio of thepolynucleic acid molecule (B) to binding moiety A is about 7 or greater.In some instances, the DAR ratio of the polynucleic acid molecule (B) tobinding moiety A is about 8 or greater. In some instances, the DAR ratioof the polynucleic acid molecule (B) to binding moiety A is about 9 orgreater. In some instances, the DAR ratio of the polynucleic acidmolecule (B) to binding moiety A is about 10 or greater. In someinstances, the DAR ratio of the polynucleic acid molecule (B) to bindingmoiety A is about 11 or greater. In some instances, the DAR ratio of thepolynucleic acid molecule (B) to binding moiety A is about 12 orgreater.

In some instances, the DAR ratio of the polynucleic acid molecule (B) tobinding moiety A (e.g., an antibody or binding fragment thereof) isabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In someinstances, the DAR ratio of the polynucleic acid molecule (B) to bindingmoiety A is about 1. In some instances, the DAR ratio of the polynucleicacid molecule (B) to binding moiety A is about 2. In some instances, theDAR ratio of the polynucleic acid molecule (B) to binding moiety A isabout 3. In some instances, the DAR ratio of the polynucleic acidmolecule (B) to binding moiety A is about 4. In some instances, the DARratio of the polynucleic acid molecule (B) to binding moiety A is about5. In some instances, the DAR ratio of the polynucleic acid molecule (B)to binding moiety A is about 6. In some instances, the DAR ratio of thepolynucleic acid molecule (B) to binding moiety A is about 7. In someinstances, the DAR ratio of the polynucleic acid molecule (B) to bindingmoiety A is about 8. In some instances, the DAR ratio of the polynucleicacid molecule (B) to binding moiety A is about 9. In some instances, theDAR ratio of the polynucleic acid molecule (B) to binding moiety A isabout 10. In some instances, the DAR ratio of the polynucleic acidmolecule (B) to binding moiety A is about 11. In some instances, the DARratio of the polynucleic acid molecule (B) to binding moiety A is about12. In some instances, the DAR ratio of the polynucleic acid molecule(B) to binding moiety A is about 13. In some instances, the DAR ratio ofthe polynucleic acid molecule (B) to binding moiety A is about 14. Insome instances, the DAR ratio of the polynucleic acid molecule (B) tobinding moiety A is about 15. In some instances, the DAR ratio of thepolynucleic acid molecule (B) to binding moiety A is about 16.

In some instances, the DAR ratio of the polynucleic acid molecule (B) tobinding moiety A is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,or 16. In some instances, the DAR ratio of the polynucleic acid molecule(B) to binding moiety A is 1. In some instances, the DAR ratio of thepolynucleic acid molecule (B) to binding moiety A is 2. In someinstances, the DAR ratio of the polynucleic acid molecule (B) to bindingmoiety A is 4. In some instances, the DAR ratio of the polynucleic acidmolecule (B) to binding moiety A is 6. In some instances, the DAR ratioof the polynucleic acid molecule (B) to binding moiety A is 8. In someinstances, the DAR ratio of the polynucleic acid molecule (B) to bindingmoiety A is 12.

In some embodiments, an antibody or its binding fragment is furthermodified using conventional techniques known in the art, for example, byusing amino acid deletion, insertion, substitution, addition, and/or byrecombination and/or any other modification (e.g. posttranslational andchemical modifications, such as glycosylation and phosphorylation) knownin the art either alone or in combination. In some instances, themodification further comprises a modification for modulating interactionwith Fc receptors. In some instances, the one or more modificationsinclude those described in, for example, International Publication No.WO97/34631, which discloses amino acid residues involved in theinteraction between the Fc domain and the FcRn receptor. Methods forintroducing such modifications in the nucleic acid sequence underlyingthe amino acid sequence of an antibody or its binding fragment is wellknown to the person skilled in the art.

In some instances, an antibody binding fragment further encompasses itsderivatives and includes polypeptide sequences containing at least oneCDR.

In some instances, the term “single-chain” as used herein means that thefirst and second domains of a bi-specific single chain construct arecovalently linked, preferably in the form of a co-linear amino acidsequence encodable by a single nucleic acid molecule.

In some instances, a bispecific single chain antibody construct relatesto a construct comprising two antibody derived binding domains. In suchembodiments, bi-specific single chain antibody construct is tandembi-scFv or diabody. In some instances, a scFv contains a VH and VLdomain connected by a linker peptide. In some instances, linkers are ofa length and sequence sufficient to ensure that each of the first andsecond domains can, independently from one another, retain theirdifferential binding specificities.

In some embodiments, binding to or interacting with as used hereindefines a binding/interaction of at least two antigen-interaction-siteswith each other. In some instances, antigen-interaction-site defines amotif of a polypeptide that shows the capacity of specific interactionwith a specific antigen or a specific group of antigens. In some cases,the binding/interaction is also understood to define a specificrecognition. In such cases, specific recognition refers to that theantibody or its binding fragment is capable of specifically interactingwith and/or binding to at least two amino acids of each of a targetmolecule. For example, specific recognition relates to the specificityof the antibody molecule, or to its ability to discriminate between thespecific regions of a target molecule. In additional instances, thespecific interaction of the antigen-interaction-site with its specificantigen results in an initiation of a signal, e.g. due to the inductionof a change of the conformation of the antigen, an oligomerization ofthe antigen, etc. In further embodiments, the binding is exemplified bythe specificity of a “key-lock-principle”. Thus in some instances,specific motifs in the amino acid sequence of theantigen-interaction-site and the antigen bind to each other as a resultof their primary, secondary or tertiary structure as well as the resultof secondary modifications of said structure. In such cases, thespecific interaction of the antigen-interaction-site with its specificantigen results as well in a simple binding of the site to the antigen.

In some instances, specific interaction further refers to a reducedcross-reactivity of the antibody or its binding fragment or a reducedoff-target effect. For example, the antibody or its binding fragmentthat bind to the polypeptide/protein of interest but do not or do notessentially bind to any of the other polypeptides are considered asspecific for the polypeptide/protein of interest. Examples for thespecific interaction of an antigen-interaction-site with a specificantigen comprise the specificity of a ligand for its receptor, forexample, the interaction of an antigenic determinant (epitope) with theantigenic binding site of an antibody.

Additional Binding Moieties

In some embodiments, the binding moiety is a plasma protein. In someinstances, the plasma protein comprises albumin. In some instances, thebinding moiety A is albumin. In some instances, albumin is conjugated byone or more of a conjugation chemistry described herein to a polynucleicacid molecule. In some instances, albumin is conjugated by nativeligation chemistry to a polynucleic acid molecule. In some instances,albumin is conjugated by lysine conjugation to a polynucleic acidmolecule.

In some instances, the binding moiety is a steroid. Exemplary steroidsinclude cholesterol, phospholipids, di- and triacylglycerols, fattyacids, hydrocarbons that are saturated, unsaturated, comprisesubstitutions, or combinations thereof. In some instances, the steroidis cholesterol. In some instances, the binding moiety is cholesterol. Insome instances, cholesterol is conjugated by one or more of aconjugation chemistry described herein to a polynucleic acid molecule.In some instances, cholesterol is conjugated by native ligationchemistry to a polynucleic acid molecule. In some instances, cholesterolis conjugated by lysine conjugation to a polynucleic acid molecule.

In some instances, the binding moiety is a polymer, including but notlimited to poly nucleic acid molecule aptamers that bind to specificsurface markers on cells. In this instance the binding moiety is apolynucleic acid that does not hybridize to a target gene or mRNA, butinstead is capable of selectively binding to a cell surface markersimilarly to an antibody binding to its specific epitope of a cellsurface marker.

In some cases, the binding moiety is a peptide. In some cases, thepeptide comprises between about 1 and about 3 kDa. In some cases, thepeptide comprises between about 1.2 and about 2.8 kDa, about 1.5 andabout 2.5 kDa, or about 1.5 and about 2 kDa. In some instances, thepeptide is a bicyclic peptide. In some cases, the bicyclic peptide is aconstrained bicyclic peptide. In some instances, the binding moiety is abicyclic peptide (e.g., bicycles from Bicycle Therapeutics).

In additional cases, the binding moiety is a small molecule. In someinstances, the small molecule is an antibody-recruiting small molecule.In some cases, the antibody-recruiting small molecule comprises atarget-binding terminus and an antibody-binding terminus, in which thetarget-binding terminus is capable of recognizing and interacting with acell surface receptor. For example, in some instances, thetarget-binding terminus comprising a glutamate urea compound enablesinteraction with PSMA, thereby, enhances an antibody interaction with acell (e.g., a cancerous cell) that expresses PSMA. In some instances, abinding moiety is a small molecule described in Zhang et al., “A remotearene-binding site on prostate specific membrane antigen revealed byantibody-recruiting small molecules,” J Am Chem Soc. 132(36):12711-12716 (2010); or McEnaney, et al., “Antibody-recruiting molecules:an emerging paradigm for engaging immune function in treating humandisease,” ACS Chem Biol. 7(7): 1139-1151 (2012).

Production of Antibodies or Binding Fragments Thereof

In some embodiments, polypeptides described herein (e.g., antibodies andits binding fragments) are produced using any method known in the art tobe useful for the synthesis of polypeptides (e.g., antibodies), inparticular, by chemical synthesis or by recombinant expression, and arepreferably produced by recombinant expression techniques.

In some instances, an antibody or its binding fragment thereof isexpressed recombinantly, and the nucleic acid encoding the antibody orits binding fragment is assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., 1994,BioTechniques 17:242), which involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligation of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

Alternatively, a nucleic acid molecule encoding an antibody isoptionally generated from a suitable source (e.g., an antibody cDNAlibrary, or cDNA library generated from any tissue or cells expressingthe immunoglobulin) by PCR amplification using synthetic primershybridizable to the 3′ and 5′ ends of the sequence or by cloning usingan oligonucleotide probe specific for the particular gene sequence.

In some instances, an antibody or its binding is optionally generated byimmunizing an animal, such as a rabbit, to generate polyclonalantibodies or, more preferably, by generating monoclonal antibodies,e.g., as described by Kohler and Milstein (1975, Nature 256:495-497) or,as described by Kozbor et al. (1983, Immunology Today 4:72) or Cole etal. (1985 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96). Alternatively, a clone encoding at least the Fabportion of the antibody is optionally obtained by screening Fabexpression libraries (e.g., as described in Huse et al., 1989, Science246:1275-1281) for clones of Fab fragments that bind the specificantigen or by screening antibody libraries (See, e.g., Clackson et al.,1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA94:4937).

In some embodiments, techniques developed for the production of“chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al.,1985, Nature 314:452-454) by splicing genes from a mouse antibodymolecule of appropriate antigen specificity together with genes from ahuman antibody molecule of appropriate biological activity are used. Achimeric antibody is a molecule in which different portions are derivedfrom different animal species, such as those having a variable regionderived from a murine monoclonal antibody and a human immunoglobulinconstant region, e.g., humanized antibodies.

In some embodiments, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,694,778; Bird, 1988, Science242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; and Ward et al., 1989, Nature 334:544-54) are adapted toproduce single chain antibodies. Single chain antibodies are formed bylinking the heavy and light chain fragments of the Fv region via anamino acid bridge, resulting in a single chain polypeptide. Techniquesfor the assembly of functional Fv fragments in E. coli are alsooptionally used (Skerra et al., 1988, Science 242:1038-1041).

In some embodiments, an expression vector comprising the nucleotidesequence of an antibody or the nucleotide sequence of an antibody istransferred to a host cell by conventional techniques (e.g.,electroporation, liposomal transfection, and calcium phosphateprecipitation), and the transfected cells are then cultured byconventional techniques to produce the antibody. In specificembodiments, the expression of the antibody is regulated by aconstitutive, an inducible or a tissue, specific promoter.

In some embodiments, a variety of host-expression vector systems isutilized to express an antibody or its binding fragment describedherein. Such host-expression systems represent vehicles by which thecoding sequences of the antibody is produced and subsequently purified,but also represent cells that are, when transformed or transfected withthe appropriate nucleotide coding sequences, express an antibody or itsbinding fragment in situ. These include, but are not limited to,microorganisms such as bacteria (e.g., E. coli and B. subtilis)transformed with recombinant bacteriophage DNA, plasmid DNA or cosmidDNA expression vectors containing an antibody or its binding fragmentcoding sequences; yeast (e.g., Saccharomyces Pichia) transformed withrecombinant yeast expression vectors containing an antibody or itsbinding fragment coding sequences; insect cell systems infected withrecombinant virus expression vectors (e.g., baculovirus) containing anantibody or its binding fragment coding sequences; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed withrecombinant plasmid expression vectors (e.g., Ti plasmid) containing anantibody or its binding fragment coding sequences; or mammalian cellsystems (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g. the adenovirus late promoter; the vaccinia virus 7.5Kpromoter).

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. In some instances, cell lines that stablyexpress an antibody are optionally engineered. Rather than usingexpression vectors that contain viral origins of replication, host cellsare transformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells are thenallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci that in turnare cloned and expanded into cell lines. This method can advantageouslybe used to engineer cell lines which express the antibody or its bindingfragments.

In some instances, a number of selection systems are used, including butnot limited to the herpes simplex virus thymidine kinase (Wigler et al.,1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase(Szybalska & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), andadenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genesare employed in tk−, hgprt− or aprt− cells, respectively. Also,antimetabolite resistance are used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981,Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418(Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95;Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan,1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev.Biochem. 62:191-217; May, 1993, TIB TECH 11(5):155-215) and hygro, whichconfers resistance to hygromycin (Santerre et al., 1984, Gene 30:147).Methods commonly known in the art of recombinant DNA technology whichcan be used are described in Ausubel et al. (eds., 1993, CurrentProtocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990,Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY;and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, CurrentProtocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin etal., 1981, J. Mol. Biol. 150:1).

In some instances, the expression levels of an antibody are increased byvector amplification (for a review, see Bebbington and Hentschel, Theuse of vectors based on gene amplification for the expression of clonedgenes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, NewYork, 1987)). When a marker in the vector system expressing an antibodyis amplifiable, an increase in the level of inhibitor present in cultureof host cell will increase the number of copies of the marker gene.Since the amplified region is associated with the nucleotide sequence ofthe antibody, production of the antibody will also increase (Crouse etal., 1983, Mol. Cell Biol. 3:257).

In some instances, any method known in the art for purification of anantibody is used, for example, by chromatography (e.g., ion exchange,affinity, particularly by affinity for the specific antigen afterProtein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins.

Polymer Conjugating Moiety

In some embodiments, a polymer moiety C is further conjugated to apolynucleic acid molecule described herein, a binding moiety describedherein, or in combinations thereof. In some instances, a polymer moietyC is conjugated a polynucleic acid molecule. In some cases, a polymermoiety C is conjugated to a binding moiety. In other cases, a polymermoiety C is conjugated to a polynucleic acid molecule-binding moietymolecule. In additional cases, a polymer moiety C is conjugated, asillustrated in FIG. 1, and as discussed under the Therapeutic MoleculePlatform section.

In some instances, the polymer moiety C is a natural or syntheticpolymer, consisting of long chains of branched or unbranched monomers,and/or cross-linked network of monomers in two or three dimensions. Insome instances, the polymer moiety C includes a polysaccharide, lignin,rubber, or polyalkylen oxide (e.g., polyethylene glycol). In someinstances, the at least one polymer moiety C includes, but is notlimited to, alpha-, omega-dihydroxylpolyethyleneglycol, biodegradablelactone-based polymer, e.g. polyacrylic acid, polylactide acid (PLA),poly(glycolic acid) (PGA), polypropylene, polystyrene, polyolefin,polyamide, polycyanoacrylate, polyimide, polyethylenterephthalat (PET,PETG), polyethylene terephthalate (PETE), polytetramethylene glycol(PTG), or polyurethane as well as mixtures thereof. As used herein, amixture refers to the use of different polymers within the same compoundas well as in reference to block copolymers. In some cases, blockcopolymers are polymers wherein at least one section of a polymer isbuild up from monomers of another polymer. In some instances, thepolymer moiety C comprises polyalkylene oxide. In some instances, thepolymer moiety C comprises PEG. In some instances, the polymer moiety Ccomprises polyethylene imide (PEI) or hydroxy ethyl starch (HES).

In some instances, C is a PEG moiety. In some instances, the PEG moietyis conjugated at the 5′ terminus of the polynucleic acid molecule whilethe binding moiety is conjugated at the 3′ terminus of the polynucleicacid molecule. In some instances, the PEG moiety is conjugated at the 3′terminus of the polynucleic acid molecule while the binding moiety isconjugated at the 5′ terminus of the polynucleic acid molecule. In someinstances, the PEG moiety is conjugated to an internal site of thepolynucleic acid molecule. In some instances, the PEG moiety, thebinding moiety, or a combination thereof, are conjugated to an internalsite of the polynucleic acid molecule. In some instances, theconjugation is a direct conjugation. In some instances, the conjugationis via native ligation.

In some embodiments, the polyalkylene oxide (e.g., PEG) is a polydispersor monodispers compound. In some instances, polydispers materialcomprises disperse distribution of different molecular weight of thematerial, characterized by mean weight (weight average) size anddispersity. In some instances, the monodisperse PEG comprises one sizeof molecules. In some embodiments, C is poly- or monodispersedpolyalkylene oxide (e.g., PEG) and the indicated molecular weightrepresents an average of the molecular weight of the polyalkylene oxide,e.g., PEG, molecules.

In some embodiments, the molecular weight of the polyalkylene oxide(e.g., PEG) is about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100,1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200,2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500, 3750,4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000,10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da.

In some embodiments, C is polyalkylene oxide (e.g., PEG) and has amolecular weight of about 200, 300, 400, 500, 600, 700, 800, 900, 1000,1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100,2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500,3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500,8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000Da. In some embodiments, C is PEG and has a molecular weight of about200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400,1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500,2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500,4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000,20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da. In someinstances, the molecular weight of C is about 200 Da. In some instances,the molecular weight of C is about 300 Da. In some instances, themolecular weight of C is about 400 Da. In some instances, the molecularweight of C is about 500 Da. In some instances, the molecular weight ofC is about 600 Da. In some instances, the molecular weight of C is about700 Da. In some instances, the molecular weight of C is about 800 Da. Insome instances, the molecular weight of C is about 900 Da. In someinstances, the molecular weight of C is about 1000 Da. In someinstances, the molecular weight of C is about 1100 Da. In someinstances, the molecular weight of C is about 1200 Da. In someinstances, the molecular weight of C is about 1300 Da. In someinstances, the molecular weight of C is about 1400 Da. In someinstances, the molecular weight of C is about 1450 Da. In someinstances, the molecular weight of C is about 1500 Da. In someinstances, the molecular weight of C is about 1600 Da. In someinstances, the molecular weight of C is about 1700 Da. In someinstances, the molecular weight of C is about 1800 Da. In someinstances, the molecular weight of C is about 1900 Da. In someinstances, the molecular weight of C is about 2000 Da. In someinstances, the molecular weight of C is about 2100 Da. In someinstances, the molecular weight of C is about 2200 Da. In someinstances, the molecular weight of C is about 2300 Da. In someinstances, the molecular weight of C is about 2400 Da. In someinstances, the molecular weight of C is about 2500 Da. In someinstances, the molecular weight of C is about 2600 Da. In someinstances, the molecular weight of C is about 2700 Da. In someinstances, the molecular weight of C is about 2800 Da. In someinstances, the molecular weight of C is about 2900 Da. In someinstances, the molecular weight of C is about 3000 Da. In someinstances, the molecular weight of C is about 3250 Da. In someinstances, the molecular weight of C is about 3350 Da. In someinstances, the molecular weight of C is about 3500 Da. In someinstances, the molecular weight of C is about 3750 Da. In someinstances, the molecular weight of C is about 4000 Da. In someinstances, the molecular weight of C is about 4250 Da. In someinstances, the molecular weight of C is about 4500 Da. In someinstances, the molecular weight of C is about 4600 Da. In someinstances, the molecular weight of C is about 4750 Da. In someinstances, the molecular weight of C is about 5000 Da. In someinstances, the molecular weight of C is about 5500 Da. In someinstances, the molecular weight of C is about 6000 Da. In someinstances, the molecular weight of C is about 6500 Da. In someinstances, the molecular weight of C is about 7000 Da. In someinstances, the molecular weight of C is about 7500 Da. In someinstances, the molecular weight of C is about 8000 Da. In someinstances, the molecular weight of C is about 10,000 Da. In someinstances, the molecular weight of C is about 12,000 Da. In someinstances, the molecular weight of C is about 20,000 Da. In someinstances, the molecular weight of C is about 35,000 Da. In someinstances, the molecular weight of C is about 40,000 Da. In someinstances, the molecular weight of C is about 50,000 Da. In someinstances, the molecular weight of C is about 60,000 Da. In someinstances, the molecular weight of C is about 100,000 Da.

In some embodiments, the polyalkylene oxide (e.g., PEG) is a discretePEG, in which the discrete PEG is a polymeric PEG comprising more thanone repeating ethylene oxide units. In some instances, a discrete PEG(dPEG) comprises from 2 to 60, from 2 to 50, or from 2 to 48 repeatingethylene oxide units. In some instances, a dPEG comprises about 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26,28, 30, 35, 40, 42, 48, 50 or more repeating ethylene oxide units. Insome instances, a dPEG comprises about 2 or more repeating ethyleneoxide units. In some instances, a dPEG comprises about 3 or morerepeating ethylene oxide units. In some instances, a dPEG comprisesabout 4 or more repeating ethylene oxide units. In some instances, adPEG comprises about 5 or more repeating ethylene oxide units. In someinstances, a dPEG comprises about 6 or more repeating ethylene oxideunits. In some instances, a dPEG comprises about 7 or more repeatingethylene oxide units. In some instances, a dPEG comprises about 8 ormore repeating ethylene oxide units. In some instances, a dPEG comprisesabout 9 or more repeating ethylene oxide units. In some instances, adPEG comprises about 10 or more repeating ethylene oxide units. In someinstances, a dPEG comprises about 11 or more repeating ethylene oxideunits. In some instances, a dPEG comprises about 12 or more repeatingethylene oxide units. In some instances, a dPEG comprises about 13 ormore repeating ethylene oxide units. In some instances, a dPEG comprisesabout 14 or more repeating ethylene oxide units. In some instances, adPEG comprises about 15 or more repeating ethylene oxide units. In someinstances, a dPEG comprises about 16 or more repeating ethylene oxideunits. In some instances, a dPEG comprises about 17 or more repeatingethylene oxide units. In some instances, a dPEG comprises about 18 ormore repeating ethylene oxide units. In some instances, a dPEG comprisesabout 19 or more repeating ethylene oxide units. In some instances, adPEG comprises about 20 or more repeating ethylene oxide units. In someinstances, a dPEG comprises about 22 or more repeating ethylene oxideunits. In some instances, a dPEG comprises about 24 or more repeatingethylene oxide units. In some instances, a dPEG comprises about 26 ormore repeating ethylene oxide units. In some instances, a dPEG comprisesabout 28 or more repeating ethylene oxide units. In some instances, adPEG comprises about 30 or more repeating ethylene oxide units. In someinstances, a dPEG comprises about 35 or more repeating ethylene oxideunits. In some instances, a dPEG comprises about 40 or more repeatingethylene oxide units. In some instances, a dPEG comprises about 42 ormore repeating ethylene oxide units. In some instances, a dPEG comprisesabout 48 or more repeating ethylene oxide units. In some instances, adPEG comprises about 50 or more repeating ethylene oxide units. In somecases, a dPEG is synthesized as a single molecular weight compound frompure (e.g., about 95%, 98%, 99%, or 99.5%) staring material in astep-wise fashion. In some cases, a dPEG has a specific molecularweight, rather than an average molecular weight. In some cases, a dPEGdescribed herein is a dPEG from Quanta Biodesign, LMD.

In some embodiments, the polymer moiety C comprises a cationic mucicacid-based polymer (cMAP). In some instances, cMPA comprises one or moresubunit of at least one repeating subunit, and the subunit structure isrepresented as Formula (III):

wherein m is independently at each occurrence 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10, preferably 4-6 or 5; and n is independently at each occurrence 1,2, 3, 4, or 5. In some embodiments, m and n are, for example, about 10.

In some instances, cMAP is further conjugated to a PEG moiety,generating a cMAP-PEG copolymer, an mPEG-cMAP-PEGm triblock polymer, ora cMAP-PEG-cMAP triblock polymer. In some instances, the PEG moiety isin a range of from about 500 Da to about 50,000 Da. In some instances,the PEG moiety is in a range of from about 500 Da to about 1000 Da,greater than 1000 Da to about 5000 Da, greater than 5000 Da to about10,000 Da, greater than 10,000 to about 25,000 Da, greater than 25,000Da to about 50,000 Da, or any combination of two or more of theseranges.

In some instances, the polymer moiety C is cMAP-PEG copolymer, anmPEG-cMAP-PEGm triblock polymer, or a cMAP-PEG-cMAP triblock polymer. Insome cases, the polymer moiety C is cMAP-PEG copolymer. In other cases,the polymer moiety C is an mPEG-cMAP-PEGm triblock polymer. Inadditional cases, the polymer moiety C is a cMAP-PEG-cMAP triblockpolymer.

In some embodiments, the polymer moiety C is conjugated to thepolynucleic acid molecule, the binding moiety, and optionally to theendosomolytic moiety as illustrated in FIG. 1A-1V.

Endosomolytic Moiety

In some embodiments, a molecule of Formula (I): A-X—B—Y—C, furthercomprises an additional conjugating moiety. In some instances, theadditional conjugating moiety is an endosomolytic moiety. In some cases,the endosomolytic moiety is a cellular compartmental release component,such as a compound capable of releasing from any of the cellularcompartments known in the art, such as the endosome, lysosome,endoplasmic reticulum (ER), golgi apparatus, microtubule, peroxisome, orother vesicular bodies with the cell. In some cases, the endosomolyticmoiety comprises an endosomolytic polypeptide, an endosomolytic polymer,an endosomolytic lipid, or an endosomolytic small molecule. In somecases, the endosomolytic moiety comprises an endosomolytic polypeptide.In other cases, the endosomolytic moiety comprises an endosomolyticpolymer.

Endosomolytic Polypeptides

In some embodiments, a molecule of Formula (I): A-X—B—Y—C, is furtherconjugated with an endosomolytic polypeptide. In some cases, theendosomolytic polypeptide is a pH-dependent membrane active peptide. Insome cases, the endosomolytic polypeptide is an amphipathic polypeptide.In additional cases, the endosomolytic polypeptide is a peptidomimetic.In some instances, the endosomolytic polypeptide comprises INF,melittin, meucin, or their respective derivatives thereof. In someinstances, the endosomolytic polypeptide comprises INF or itsderivatives thereof. In other cases, the endosomolytic polypeptidecomprises melittin or its derivatives thereof. In additional cases, theendosomolytic polypeptide comprises meucin or its derivatives thereof.

In some instances, INF7 is a 24 residue polypeptide those sequencecomprises CGIFGEIEELIEEGLENLIDWGNA (SEQ ID NO: 2055), orGLFEAIEGFIENGWEGMIDGWYGC (SEQ ID NO: 2056). In some instances, INF7 orits derivatives comprise a sequence of: GLFEAIEGFIENGWEGMIWDYGSGSCG (SEQID NO: 2057), GLFEAIEGFIENGWEGMIDG WYG-(PEG)6-NH2 (SEQ ID NO: 2058), orGLFEAIEGFIENGWEGMIWDYG-SGSC-K(GalNAc)2 (SEQ ID NO: 2059).

In some cases, melittin is a 26 residue polypeptide those sequencecomprises CLIGAILKVLATGLPTLISWIKNKRKQ (SEQ ID NO: 2060), orGIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 2061). In some instances,melittin comprises a polypeptide sequence as described in U.S. Pat. No.8,501,930.

In some instances, meucin is an antimicrobial peptide (AMP) derived fromthe venom gland of the scorpion Mesobuthus eupeus. In some instances,meucin comprises of meucin-13 those sequence comprises IFGAIAGLLKNIF-NH2(SEQ ID NO: 2062) and meucin-18 those sequence comprisesFFGHLFKLATKIIPSLFQ (SEQ ID NO: 2063).

In some instances, the endosomolytic polypeptide comprises a polypeptidein which its sequence is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99%sequence identity to INF7 or its derivatives thereof, melittin or itsderivatives thereof, or meucin or its derivatives thereof. In someinstances, the endosomolytic moiety comprises INF7 or its derivativesthereof, melittin or its derivatives thereof, or meucin or itsderivatives thereof.

In some instances, the endosomolytic moiety is INF7 or its derivativesthereof. In some cases, the endosomolytic moiety comprises a polypeptidehaving at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2055-2059. Insome cases, the endosomolytic moiety comprises a polypeptide having atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to SEQ ID NO: 2055. In some cases, theendosomolytic moiety comprises a polypeptide having at least 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to SEQ ID NO: 2056-2059. In some cases, theendosomolytic moiety comprises SEQ ID NO: 2055. In some cases, theendosomolytic moiety comprises SEQ ID NO: 2056-2059. In some cases, theendosomolytic moiety consists of SEQ ID NO: 2055. In some cases, theendosomolytic moiety consists of SEQ ID NO: 2056-2059.

In some instances, the endosomolytic moiety is melittin or itsderivatives thereof. In some cases, the endosomolytic moiety comprises apolypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2060or 2061. In some cases, the endosomolytic moiety comprises a polypeptidehaving at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2060. In somecases, the endosomolytic moiety comprises a polypeptide having at least50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity to SEQ ID NO: 2061. In some cases, theendosomolytic moiety comprises SEQ ID NO: 2060. In some cases, theendosomolytic moiety comprises SEQ ID NO: 2061. In some cases, theendosomolytic moiety consists of SEQ ID NO: 2060. In some cases, theendosomolytic moiety consists of SEQ ID NO: 2061.

In some instances, the endosomolytic moiety is meucin or its derivativesthereof. In some cases, the endosomolytic moiety comprises a polypeptidehaving at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2062 or 2063. Insome cases, the endosomolytic moiety comprises a polypeptide having atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to SEQ ID NO: 2062. In some cases, theendosomolytic moiety comprises a polypeptide having at least 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to SEQ ID NO: 2063. In some cases, the endosomolyticmoiety comprises SEQ ID NO: 2062. In some cases, the endosomolyticmoiety comprises SEQ ID NO: 2063. In some cases, the endosomolyticmoiety consists of SEQ ID NO: 2062. In some cases, the endosomolyticmoiety consists of SEQ ID NO: 2063.

In some instances, the endosomolytic moiety comprises a sequence asillustrated in Table 62.

TABLE 62 SEQ ID Name Origin Amino Acid Sequence NO: Type Pep-1 NLS fromSimian Virus KETWWETWWTEWSQPKKKRKV 2064 Primary 40 large antigen andamphipathic Reverse transcriptase of HIV pVEC VE-cadherinLLIILRRRRIRKQAHAHSK 2065 Primary amphipathic VT5 Synthetic peptideDPKGDPKGVTVTVTVTVTGKGDP 2066 β-sheet KPD amphipathic C105Y 1-antitrypsinCSIPPEVKFNKPFVYLI 2067 — Transportan Galanin and mastoparanGWTLNSAGYLLGKINLKALAALA 2068 Primary KKIL amphipathic TP10 Galanin andmastoparan AGYLLGKINLKALAALAKKIL 2069 Primary amphipathic MPG Ahydrofobic domain GALFLGFLGAAGSTMGA 2070 β-sheet from the fusionamphipathic sequence of HIV gp41 and NLS of SV40 T antigen gH625Glycoprotein gH of HGLASTLTRWAHYNALIRAF 2071 Secondary HSV type Iamphipathic α-helical CADY PPTG1 peptide GLWRALWRLLRSLWRLLWRA 2072Secondary amphipathic α-helical GALA Synthetic peptideWEAALAEALAEALAEHLAEALAE 2073 Secondary ALEALAA amphipathic α-helical INFInfluenza HA2 fusion GLFEAIEGFIENGWEGMIDGWYGC 2074 Secondary peptideamphipathic α-helical/ pH- dependent membrane active peptide HA2E5-Influenza HA2 subunit GLFGAIAGFIENGWEGMIDGWYG 2075 Secondary TAT ofinfluenza virus X31 amphipathic strain fusion peptide α-helical/ pH-dependent membrane active peptide HA2- Influenza HA2 subunitGLFGAIAGFIENGWEGMIDGRQIKI 2076 pH- penetratin of influenza virus X31WFQNRRMKW dependent strain fusion peptide KK-amide membrane activepeptide HA-K4 Influenza HA2 subunit GLFGAIAGFIENGWEGMIDG- 2077 pH- ofinfluenza virus X31 SSKKKK dependent strain fusion peptide membraneactive peptide HA2E4 Influenza HA2 subunit GLFEAIAGFIENGWEGMIDGGGYC 2078pH- of influenza virus X31 dependent strain fusion peptide membraneactive peptide H5WYG HA2 analogue GLFHAIAHFIHGGWH 2079 pH- GLIHGWYGdependent membrane active peptide GALA- INF3 fusion peptideGLFEAIEGFIENGWEGLAEALAEAL 2080 pH- INF3- EALAA- dependent (PEG)6-NH(PEG)6-NH2 membrane active peptide CM18- Cecropin-A-Melittin₂₋₁₂KWKLFKKIGAVLKVLTTG- 2081 pH- TAT11 (CM₁₈) fusion peptide YGRKKRRQRRRdependent membrane active peptide

In some cases, the endosomolytic moiety comprises a Bak BH3 polypeptidewhich induces apoptosis through antagonization of suppressor targetssuch as Bcl-2 and/or Bcl-x_(L). In some instances, the endosomolyticmoiety comprises a Bak BH3 polypeptide described in Albarran, et al.,“Efficient intracellular delivery of a pro-apoptotic peptide with apH-responsive carrier,” Reactive & Functional Polymers 71: 261-265(2011).

In some instances, the endosomolytic moiety comprises a polypeptide(e.g., a cell-penetrating polypeptide) as described in PCT PublicationNos. WO2013/166155 or WO2015/069587.

Endosomolytic Polymers

In some embodiments, a molecule of Formula (I): A-X—B—Y—C, is furtherconjugated with an endosomolytic polymer. As used herein, anendosomolytic polymer comprises a linear, a branched network, a star, acomb, or a ladder type of polymer. In some instances, an endosomolyticpolymer is a homopolymer or a copolymer comprising two ro more differenttypes of monomers. In some cases, an endosomolytic polymer is apolycation polymer. In other cases, an endosomolytic polymer is apolyanion polymer.

In some instances, a polycation polymer comprises monomer units that arecharge positive, charge neutral, or charge negative, with a net chargebeing positive. In other cases, a polycation polymer comprises anon-polymeric molecule that contains two or more positive charges.Exemplary cationic polymers include, but are not limited to,poly(L-lysine) (PLL), poly(L-arginine) (PLA), polyethyleneimine (PEI),poly[α-(4-aminobutyl)-L-glycolic acid] (PAGA), 2-(dimethylamino)ethylmethacrylate (DMAEMA), or N,N-Diethy laminoethyl Methacrylate (DEAEMA).

In some cases, a polyanion polymer comprises monomer units that arecharge positive, charge neutral, or charge negative, with a net chargebeing negative. In other cases, a polyanion polymer comprises anon-polymeric molecule that contains two or more negative charges.Exemplary anionic polymers include p(alkylacrylates) (e.g., poly(propylacrylic acid) (PPAA)) or poly(N-isopropylacrylamide) (NIPAM). Additionalexamples include PP75, a L-phenylalanine-poly(L-lysine isophthalamide)polymer described in Khormaee, et al., “Edosomolytic anionic polymer forthe cytoplasmic delivery of siRNAs in localized in vivo applications,”Advanced Functional Materials 23: 565-574 (2013).

In some embodiments, an endosomolytic polymer described herein is apH-responsive endosomolytic polymer. A pH-responsive polymer comprises apolymer that increases in size (swell) or collapses depending on the pHof the environment. Polyacrylic acid and chitosan are examples ofpH-responsive polymers.

In some instances, an endosomolytic moiety described herein is amembrane-disruptive polymer. In some cases, the membrane-disruptivepolymer comprises a cationic polymer, a neutral or hydrophobic polymer,or an anionic polymer. In some instances, the membrane-disruptivepolymer is a hydrophilic polymer.

In some instances, an endosomolytic moiety described herein is apH-responsive membrane-disruptive polymer. Exemplary pH-responsivemembrane-disruptive polymers include p(alkylacrylic acids),poly(N-isopropylacrylamide) (NIPAM) copolymers, succinylatedp(glycidols), and p(β-malic acid) polymers.

In some instances, p(alkylacrylic acids) include poly(propylacrylicacid) (polyPAA), poly(methacrylic acid) (PMAA), poly(ethylacrylic acid)(PEAA), and poly(propyl acrylic acid) (PPAA). In some instances, ap(alkylacrylic acid) include a p(alkylacrylic acid) described in Jones,et al., Biochemistry Journal 372: 65-75 (2003).

In some embodiments, a pH-responsive membrane-disruptive polymercomprises p(butyl acrylate-co-methacrylic acid). (see Bulmus, et al.,Journal of Controlled Release 93: 105-120 (2003); and Yessine, et al.,Biochimica et Biophysica Acta 1613: 28-38 (2003))

In some embodiments, a pH-responsive membrane-disruptive polymercomprises p(styrene-alt-maleic anhydride). (see Henry, et al.,Biomacromolecules 7: 2407-2414 (2006))

In some embodiments, a pH-responsive membrane-disruptive polymercomprises pyridyldisulfide acrylate (PDSA) polymers such aspoly(MAA-co-PDSA), poly(EAA-co-PDSA), poly(PAA-co-PDSA),poly(MAA-co-BA-co-PDSA), poly(EAA-co-BA-co-PDSA), orpoly(PAA-co-BA-co-PDSA) polymers. (see El-Sayed, et al., “Rationaldesign of composition and activity correlations for pH-responsive andglutathione-reactive polymer therapeutics,” Journal of ControlledRelease 104: 417-427 (2005); or Flanary et al., “Antigen delivery withpoly(propylacrylic acid) conjugation enhanced MHC-1 presentation andT-cell activation,” Bioconjugate Chem. 20: 241-248 (2009))

In some embodiments, a pH-responsive membrane-disruptive polymercomprises a lytic polymer comprising the base structure of:

In some instances, an endosomolytic moiety described herein is furtherconjugated to an additional conjugate, e.g., a polymer (e.g., PEG), or amodified polymer (e.g., cholesterol-modified polymer).

In some instances, the additional conjugate comprises a detergent (e.g.,Triton X-100). In some instances, an endosomolytic moiety describedherein comprises a polymer (e.g., a poly(amidoamine)) conjugated with adetergent (e.g., Triton X-100). In some instances, an endosomolyticmoiety described herein comprises poly(amidoamine)-Triton X-100conjugate (Duncan, et al., “A polymer-Triton X-100 conjugate capable ofpH-dependent red blood cell lysis: a model system illustrating thepossibility of drug delivery within acidic intracellular compartments,”Journal of Drug Targeting 2: 341-347 (1994)).

Endosomolytic Lipids

In some embodiments, the endosomolytic moiety is a lipid (e.g., afusogenic lipid). In some embodiments, a molecule of Formula (I):A-X—B—Y—C, is further conjugated with an endosomolytic lipid (e.g.,fusogenic lipid). Exemplary fusogenic lipids include1,2-dileoyl-sn-3-phosphoethanolamine (DOPE), phosphatidylethanolamine(POPE), palmitoyloleoylphosphatidylcholine (POPC),(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (Di-Lin),N-methyl(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)methanamine(DLin-k-DMA) andN-methyl-2-(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)ethanamine(XTC).

In some instances, an endosomolytic moiety is a lipid (e.g., a fusogeniclipid) described in PCT Publication No. WO09/126,933.

Endosomolytic Small Molecules

In some embodiments, the endosomolytic moiety is a small molecule. Insome embodiments, a molecule of Formula (I): A-X—B—Y—C, is furtherconjugated with an endosomolytic small molecule. Exemplary smallmolecules suitable as endosomolytic moieties include, but are notlimited to, quinine, chloroquine, hydroxychloroquines, amodiaquins(carnoquines), amopyroquines, primaquines, mefloquines, nivaquines,halofantrines, quinone imines, or a combination thereof. In someinstances, quinoline endosomolytic moieties include, but are not limitedto, 7-chloro-4-(4-diethylamino-1-methylbutyl-amino)quinoline(chloroquine);7-chloro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutyl-amino)quinoline(hydroxychloroquine);7-fluoro-4-(4-diethylamino-1-methylbutyl-amino)quinoline;4-(4-diethylamino-1-methylbutylamino) quinoline;7-hydroxy-4-(4-diethyl-amino-1-methylbutylamino)quinoline;7-chloro-4-(4-diethylamino-1-butylamino)quinoline(desmethylchloroquine);7-fluoro-4-(4-diethylamino-1-butylamino)quinoline);4-(4-diethyl-amino-1-butylamino)quinoline;7-hydroxy-4-(4-diethylamino-1-butylamino)quinoline;7-chloro-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline;7-fluoro-4-(1-carboxy-4-diethyl-amino-1-butylamino)quinoline;4-(1-carboxy-4-diethylamino-1-butylamino) quinoline;7-hydroxy-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline;7-chloro-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline;7-fluoro-4-(1-carboxy-4-diethyl-amino-1-methylbutylamino)quinoline;4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline;7-hydroxy-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline;7-fluoro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;4-(4-ethyl-(2-hydroxy-ethyl)-amino-1-methylbutylamino-)quinoline;7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;hydroxychloroquine phosphate;7-chloro-4-(4-ethyl-(2-hydroxyethyl-1)-amino-1-butylamino)quinoline(desmethylhydroxychloroquine);7-fluoro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino) quinoline;7-chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;7-fluoro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;7-hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;7-chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;7-fluoro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;7-hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;8-[(4-aminopentyl)amino-6-methoxydihydrochloride quinoline;1-acetyl-1,2,3,4-tetrahydroquinoline;8-[(4-aminopentyl)amino]-6-methoxyquinoline dihydrochloride;1-butyryl-1,2,3,4-tetrahydroquinoline;3-chloro-4-(4-hydroxy-alpha,alpha′-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline,4-[(4-diethyl-amino)-1-methylbutyl-amino]-6-methoxyquinoline;3-fluoro-4-(4-hydroxy-alpha,alpha′-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline,4-[(4-diethylamino)-1-methylbutyl-amino]-6-methoxyquinoline;4-(4-hydroxy-alpha,alpha′-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline;4-[(4-diethylamino)-1-methylbutyl-amino]-6-methoxyquinoline;3,4-dihydro-1-(2H)-quinolinecarboxyaldehyde; 1,1′-pentamethylenediquinoleinium diiodide; 8-quinolinol sulfate and amino, aldehyde,carboxylic, hydroxyl, halogen, keto, sulfhydryl and vinyl derivatives oranalogs thereof. In some instances, an endosomolytic moiety is a smallmolecule described in Naisbitt et al (1997, J Pharmacol Exp Therapy280:884-893) and in U.S. Pat. No. 5,736,557.

Formula (I) Molecule—Endosomolytic Moiety Conjugates

In some embodiments, one or more endosomolytic moieties are conjugatedto a molecule comprising at least one binding moiety, at least onepolynucleotide, at least one polymer, or any combinations thereof. Insome instances, the endosomolytic moiety is conjugated according toFormula (II):(A-X—B—Y—C_(c))-L-D   Formula IIwherein,

A is a binding moiety;

B is a polynucleotide;

C is a polymer;

X is a bond or first linker;

Y is a bond or second linker;

L is a bond or third linker;

D is an endosomolytic moiety; and

c is an integer between 0 and 1; and

wherein the polynucleotide comprises at least one 2′ modifiednucleotide, at least one modified internucleotide linkage, or at leastone inverted abasic moiety; and D is conjugated anywhere on A, B, or C.

In some embodiments, A and C are not attached to B at the same terminus.

In some embodiments, the at least one 2′ modified nucleotide comprises2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy,T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido(2′-O-NMA) modified nucleotide. In some instances, the at least one 2′modified nucleotide comprises locked nucleic acid (LNA) or ethylenenucleic acid (ENA). In some cases, the at least one modifiedinternucleotide linkage comprises a phosphorothioate linkage or aphosphorodithioate linkage. In some embodiments, the polynucleotidecomprises a first polynucleotide and a second polynucleotide hybridizedto the first polynucleotide to form a double-stranded polynucleic acidmolecule. In some instances, the second polynucleotide comprises atleast one modification. In some cases, the first polynucleotide and thesecond polynucleotide are RNA molecules. In some cases, the firstpolynucleotide and the second polynucleotide are siRNA molecules. Insome embodiments, X, Y, and L are independently a bond or anon-polymeric linker group. In some instances, A is an antibody orbinding fragment thereof. In some instances, the antibody or bindingfragment thereof comprises a humanized antibody or binding fragmentthereof, chimeric antibody or binding fragment thereof, monoclonalantibody or binding fragment thereof, monovalent Fab′, divalent Fab2,single-chain variable fragment (scFv), diabody, minibody, nanobody,single-domain antibody (sdAb), or camelid antibody or binding fragmentthereof. In some cases, C is polyethylene glycol.

In some instances, the endosomolytic moiety comprises a polypeptide, apolymer, a lipid, or a small molecule. In some instances, theendosomolytic moiety is an endosomolytic polypeptide. In some cases, theendosomolytic moiety is an endosomolytic polymer. In other cases, theendosomolytic moiety is an endosomolytic lipid. In additional cases, theendosomolytic moiety is an endosomolytic small molecule.

In some instances, the endosomolytic moiety is INF7 or its derivativesthereof. In some cases, the endosomolytic moiety comprises a polypeptidehaving at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2055. In somecases, the endosomolytic moiety comprises SEQ ID NO: 2055. In somecases, the endosomolytic moiety consists of SEQ ID NO: 2055.

In some instances, the endosomolytic moiety is melittin or itsderivatives thereof. In some cases, the endosomolytic moiety comprises apolypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2060.In some cases, the endosomolytic moiety comprises SEQ ID NO: 2060. Insome cases, the endosomolytic moiety consists of SEQ ID NO: 2060.

In some instances, the endosomolytic moiety is a sequence as illustratedin Table 62.

In additional cases, the endosomolytic moiety is an endosomolyticpolymer, such as for example, a pH-responsive endosomolytic polymer, amembrane-disruptive polymer, a polycation polymer, a polyanion polymer,a pH-responsive membrane-disruptive polymer, or a combination thereof.In additional cases, the endosomolytic moiety comprises a p(alkylacrylicacid) polymer, a p(butyl acrylate-co-methacrylic acid) polymer, ap(styrene-alt-maleic anhydride) polymer, a pyridyldisulfide acrylate(PDSA) polymer, a polymer-PEG conjugate, a polymer-detergent conjugate,or a combination thereof.

In some embodiments, the endosomolytic moiety conjugate is according toFormula (IIa):D-L-A-X—B—Y—C_(c)   Formula IIawherein,

A is a binding moiety;

B is a polynucleotide;

C is a polymer;

X is a bond or first linker;

Y is a bond or second linker;

L is a bond or third linker;

D is an endosomolytic moiety; and

c is an integer of 1; and

wherein the polynucleotide comprises at least one 2′ modifiednucleotide, at least one modified internucleotide linkage, or at leastone inverted abasic moiety.

In some embodiments, A and C are not attached to B at the same terminus.

In some embodiments, the at least one 2′ modified nucleotide comprises2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy,T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido(2′-O-NMA) modified nucleotide. In some instances, the at least one 2′modified nucleotide comprises locked nucleic acid (LNA) or ethylenenucleic acid (ENA). In some cases, the at least one modifiedinternucleotide linkage comprises a phosphorothioate linkage or aphosphorodithioate linkage. In some embodiments, the polynucleotidecomprises a first polynucleotide and a second polynucleotide hybridizedto the first polynucleotide to form a double-stranded polynucleic acidmolecule. In some instances, the second polynucleotide comprises atleast one modification. In some cases, the first polynucleotide and thesecond polynucleotide are RNA molecules. In some cases, the firstpolynucleotide and the second polynucleotide are siRNA molecules. Insome embodiments, X, Y, and L are independently a bond or anon-polymeric linker group. In some instances, A is an antibody orbinding fragment thereof. In some instances, the antibody or bindingfragment thereof comprises a humanized antibody or binding fragmentthereof, chimeric antibody or binding fragment thereof, monoclonalantibody or binding fragment thereof, monovalent Fab′, divalent Fab2,single-chain variable fragment (scFv), diabody, minibody, nanobody,single-domain antibody (sdAb), or camelid antibody or binding fragmentthereof. In some cases, C is polyethylene glycol.

In some instances, the endosomolytic moiety comprises a polypeptide, apolymer, a lipid, or a small molecule. In some instances, theendosomolytic moiety is an endosomolytic polypeptide. In some cases, theendosomolytic moiety is an endosomolytic polymer. In other cases, theendosomolytic moiety is an endosomolytic lipid. In additional cases, theendosomolytic moiety is an endosomolytic small molecule.

In some instances, the endosomolytic moiety is INF7 or its derivativesthereof. In some cases, the endosomolytic moiety comprises a polypeptidehaving at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2055. In somecases, the endosomolytic moiety comprises SEQ ID NO: 2055. In somecases, the endosomolytic moiety consists of SEQ ID NO: 2055.

In some instances, the endosomolytic moiety is melittin or itsderivatives thereof. In some cases, the endosomolytic moiety comprises apolypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2060.In some cases, the endosomolytic moiety comprises SEQ ID NO: 2060. Insome cases, the endosomolytic moiety consists of SEQ ID NO: 2060.

In some instances, the endosomolytic moiety is a sequence as illustratedin Table 62.

In additional cases, the endosomolytic moiety is an endosomolyticpolymer, such as for example, a pH-responsive endosomolytic polymer, amembrane-disruptive polymer, a polycation polymer, a polyanion polymer,a pH-responsive membrane-disruptive polymer, or a combination thereof.In additional cases, the endosomolytic moiety comprises a p(alkylacrylicacid) polymer, a p(butyl acrylate-co-methacrylic acid) polymer, ap(styrene-alt-maleic anhydride) polymer, a pyridyldisulfide acrylate(PDSA) polymer, a polymer-PEG conjugate, a polymer-detergent conjugate,or a combination thereof.

In some instances, the endosomolytic moiety conjugate is according toFormula (IIb):A-X—B-L-D   Formula IIbwherein,

A is a binding moiety;

B is a polynucleotide;

X is a bond or first linker;

L is a bond or third linker; and

D is an endosomolytic moiety; and

wherein the polynucleotide comprises at least one 2′ modifiednucleotide, at least one modified internucleotide linkage, or at leastone inverted abasic moiety.

In some embodiments, A and C are not attached to B at the same terminus.

In some embodiments, the at least one 2′ modified nucleotide comprises2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy,T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido(2′-O-NMA) modified nucleotide. In some instances, the at least one 2′modified nucleotide comprises locked nucleic acid (LNA) or ethylenenucleic acid (ENA). In some cases, the at least one modifiedinternucleotide linkage comprises a phosphorothioate linkage or aphosphorodithioate linkage. In some embodiments, the polynucleotidecomprises a first polynucleotide and a second polynucleotide hybridizedto the first polynucleotide to form a double-stranded polynucleic acidmolecule. In some instances, the second polynucleotide comprises atleast one modification. In some cases, the first polynucleotide and thesecond polynucleotide are RNA molecules. In some cases, the firstpolynucleotide and the second polynucleotide are siRNA molecules. Insome embodiments, X and L are independently a bond or a non-polymericlinker group. In some instances, A is an antibody or binding fragmentthereof. In some instances, the antibody or binding fragment thereofcomprises a humanized antibody or binding fragment thereof, chimericantibody or binding fragment thereof, monoclonal antibody or bindingfragment thereof, monovalent Fab′, divalent Fab2, single-chain variablefragment (scFv), diabody, minibody, nanobody, single-domain antibody(sdAb), or camelid antibody or binding fragment thereof. In some cases,C is polyethylene glycol.

In some instances, the endosomolytic moiety comprises a polypeptide, apolymer, a lipid, or a small molecule. In some instances, theendosomolytic moiety is an endosomolytic polypeptide. In some cases, theendosomolytic moiety is an endosomolytic polymer. In other cases, theendosomolytic moiety is an endosomolytic lipid. In additional cases, theendosomolytic moiety is an endosomolytic small molecule.

In some instances, the endosomolytic moiety is INF7 or its derivativesthereof. In some cases, the endosomolytic moiety comprises a polypeptidehaving at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2055. In somecases, the endosomolytic moiety comprises SEQ ID NO: 2055. In somecases, the endosomolytic moiety consists of SEQ ID NO: 2055.

In some instances, the endosomolytic moiety is melittin or itsderivatives thereof. In some cases, the endosomolytic moiety comprises apolypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2060.In some cases, the endosomolytic moiety comprises SEQ ID NO: 2060. Insome cases, the endosomolytic moiety consists of SEQ ID NO: 2060.

In some instances, the endosomolytic moiety is a sequence as illustratedin Table 62.

In additional cases, the endosomolytic moiety is an endosomolyticpolymer, such as for example, a pH-responsive endosomolytic polymer, amembrane-disruptive polymer, a polycation polymer, a polyanion polymer,a pH-responsive membrane-disruptive polymer, or a combination thereof.In additional cases, the endosomolytic moiety comprises a p(alkylacrylicacid) polymer, a p(butyl acrylate-co-methacrylic acid) polymer, ap(styrene-alt-maleic anhydride) polymer, a pyridyldisulfide acrylate(PDSA) polymer, a polymer-PEG conjugate, a polymer-detergent conjugate,or a combination thereof.

In some instances, the endosomolytic moiety conjugate is according toFormula (IIc):A-X—B—Y—C_(c)-L-D   Formula IIcwherein,

A is a binding moiety;

B is a polynucleotide;

C is a polymer;

X is a bond or first linker;

Y is a bond or second linker;

L is a bond or third linker;

D is an endosomolytic moiety; and

c is an integer of 1; and

wherein the polynucleotide comprises at least one 2′ modifiednucleotide, at least one modified internucleotide linkage, or at leastone inverted abasic moiety.

In some embodiments, A and C are not attached to B at the same terminus.

In some embodiments, the at least one 2′ modified nucleotide comprises2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy,T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido(2′-O-NMA) modified nucleotide. In some instances, the at least one 2′modified nucleotide comprises locked nucleic acid (LNA) or ethylenenucleic acid (ENA). In some cases, the at least one modifiedinternucleotide linkage comprises a phosphorothioate linkage or aphosphorodithioate linkage. In some embodiments, the polynucleotidecomprises a first polynucleotide and a second polynucleotide hybridizedto the first polynucleotide to form a double-stranded polynucleic acidmolecule. In some instances, the second polynucleotide comprises atleast one modification. In some cases, the first polynucleotide and thesecond polynucleotide are RNA molecules. In some cases, the firstpolynucleotide and the second polynucleotide are siRNA molecules. Insome embodiments, X, Y, and L are independently a bond or anon-polymeric linker group. In some instances, A is an antibody orbinding fragment thereof. In some instances, the antibody or bindingfragment thereof comprises a humanized antibody or binding fragmentthereof, chimeric antibody or binding fragment thereof, monoclonalantibody or binding fragment thereof, monovalent Fab′, divalent Fab2,single-chain variable fragment (scFv), diabody, minibody, nanobody,single-domain antibody (sdAb), or camelid antibody or binding fragmentthereof. In some cases, C is polyethylene glycol.

In some instances, the endosomolytic moiety comprises a polypeptide, apolymer, a lipid, or a small molecule. In some instances, theendosomolytic moiety is an endosomolytic polypeptide. In some cases, theendosomolytic moiety is an endosomolytic polymer. In other cases, theendosomolytic moiety is an endosomolytic lipid. In additional cases, theendosomolytic moiety is an endosomolytic small molecule.

In some instances, the endosomolytic moiety is INF7 or its derivativesthereof. In some cases, the endosomolytic moiety comprises a polypeptidehaving at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2055. In somecases, the endosomolytic moiety comprises SEQ ID NO: 2055. In somecases, the endosomolytic moiety consists of SEQ ID NO: 2055.

In some instances, the endosomolytic moiety is melittin or itsderivatives thereof. In some cases, the endosomolytic moiety comprises apolypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2060.In some cases, the endosomolytic moiety comprises SEQ ID NO: 2060. Insome cases, the endosomolytic moiety consists of SEQ ID NO: 2060.

In some instances, the endosomolytic moiety is a sequence as illustratedin Table 62.

In additional cases, the endosomolytic moiety is an endosomolyticpolymer, such as for example, a pH-responsive endosomolytic polymer, amembrane-disruptive polymer, a polycation polymer, a polyanion polymer,a pH-responsive membrane-disruptive polymer, or a combination thereof.In additional cases, the endosomolytic moiety comprises a p(alkylacrylicacid) polymer, a p(butyl acrylate-co-methacrylic acid) polymer, ap(styrene-alt-maleic anhydride) polymer, a pyridyldisulfide acrylate(PDSA) polymer, a polymer-PEG conjugate, a polymer-detergent conjugate,or a combination thereof.

In some instances, the endosomolytic moiety conjugate is according toFormula (IId):A-L-D-X—B—Y—C_(c)   Formula IIdwherein,

A is a binding moiety;

B is a polynucleotide;

C is a polymer;

X is a bond or first linker;

Y is a bond or second linker;

L is a bond or third linker;

D is an endosomolytic moiety; and

c is an integer of 1; and

wherein the polynucleotide comprises at least one 2′ modifiednucleotide, at least one modified internucleotide linkage, or at leastone inverted abasic moiety.

In some embodiments, A and C are not attached to B at the same terminus.

In some embodiments, the at least one 2′ modified nucleotide comprises2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy,T-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido(2′-O-NMA) modified nucleotide. In some instances, the at least one 2′modified nucleotide comprises locked nucleic acid (LNA) or ethylenenucleic acid (ENA). In some cases, the at least one modifiedinternucleotide linkage comprises a phosphorothioate linkage or aphosphorodithioate linkage. In some embodiments, the polynucleotidecomprises a first polynucleotide and a second polynucleotide hybridizedto the first polynucleotide to form a double-stranded polynucleic acidmolecule. In some instances, the second polynucleotide comprises atleast one modification. In some cases, the first polynucleotide and thesecond polynucleotide are RNA molecules. In some cases, the firstpolynucleotide and the second polynucleotide are siRNA molecules. Insome embodiments, X, Y, and L are independently a bond or anon-polymeric linker group. In some instances, A is an antibody orbinding fragment thereof. In some instances, the antibody or bindingfragment thereof comprises a humanized antibody or binding fragmentthereof, chimeric antibody or binding fragment thereof, monoclonalantibody or binding fragment thereof, monovalent Fab′, divalent Fab2,single-chain variable fragment (scFv), diabody, minibody, nanobody,single-domain antibody (sdAb), or camelid antibody or binding fragmentthereof. In some cases, C is polyethylene glycol.

In some instances, the endosomolytic moiety comprises a polypeptide, apolymer, a lipid, or a small molecule. In some instances, theendosomolytic moiety is an endosomolytic polypeptide. In some cases, theendosomolytic moiety is an endosomolytic polymer. In other cases, theendosomolytic moiety is an endosomolytic lipid. In additional cases, theendosomolytic moiety is an endosomolytic small molecule.

In some instances, the endosomolytic moiety is INF7 or its derivativesthereof. In some cases, the endosomolytic moiety comprises a polypeptidehaving at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2055. In somecases, the endosomolytic moiety comprises SEQ ID NO: 2055. In somecases, the endosomolytic moiety consists of SEQ ID NO: 2055.

In some instances, the endosomolytic moiety is melittin or itsderivatives thereof. In some cases, the endosomolytic moiety comprises apolypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 2060.In some cases, the endosomolytic moiety comprises SEQ ID NO: 2060. Insome cases, the endosomolytic moiety consists of SEQ ID NO: 2060.

In some instances, the endosomolytic moiety is a sequence as illustratedin Table 62.

In additional cases, the endosomolytic moiety is an endosomolyticpolymer, such as for example, a pH-responsive endosomolytic polymer, amembrane-disruptive polymer, a polycation polymer, a polyanion polymer,a pH-responsive membrane-disruptive polymer, or a combination thereof.In additional cases, the endosomolytic moiety comprises a p(alkylacrylicacid) polymer, a p(butyl acrylate-co-methacrylic acid) polymer, ap(styrene-alt-maleic anhydride) polymer, a pyridyldisulfide acrylate(PDSA) polymer, a polymer-PEG conjugate, a polymer-detergent conjugate,or a combination thereof.

Linkers

In some embodiments, a linker described herein is a cleavable linker ora non-cleavable linker. In some instances, the linker is a cleavablelinker. In some instances, the linker is an acid cleavable linker. Insome instances, the linker is a non-cleavable linker. In some instances,the linker includes a C₁-C₆ alkyl group (e.g., a C₅, C₄, C₃, C₂, or C₁alkyl group). In some instances, the linker includes homobifunctionalcross linkers, heterobifunctional cross linkers, and the like. In someinstances, the liker is a traceless linker (or a zero-length linker). Insome instances, the linker is a non-polymeric linker. In some cases, thelinker is a non-peptide linker or a linker that does not contain anamino acid residue.

In some instances, the linker comprises a homobifuctional linker.Exemplary homobifuctional linkers include, but are not limited to,Lomant's reagent dithiobis (succinimidylpropionate) DSP,3′3′-dithiobis(sulfosuccinimidyl proprionate (DTSSP), disuccinimidylsuberate (DSS), bis(sulfosuccinimidyl)suberate (BS), disuccinimidyltartrate (DST), disulfosuccinimidyl tartrate (sulfo DST), ethyleneglycobis(succinimidylsuccinate) (EGS), disuccinimidyl glutarate (DSG),N,N′-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA),dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS),dimethyl-3,3′-dithiobispropionimidate (DTBP),1,4-di-3′-(2′-pyridyldithio)propionamido)butane (DPDPB),bismaleimidohexane (BMH), aryl halide-containing compound (DFDNB), suchas e.g. 1,5-difluoro-2,4-dinitrobenzene or1,3-difluoro-4,6-dinitrobenzene, 4,4′-difluoro-3,3′-dinitrophenylsulfone(DFDNPS), bis-[β-(4-azidosalicylamido)ethyl]disulfide (BASED),formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipicacid dihydrazide, carbohydrazide, o-toluidine, 3,3′-dimethylbenzidine,benzidine, α,α′-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid,N,N′-ethylene-bis(iodoacetamide), orN,N′-hexamethylene-bis(iodoacetamide).

In some embodiments, the linker comprises a heterobifunctional linker.Exemplary heterobifunctional linker include, but are not limited to,amine-reactive and sulfhydryl cross-linkers such as N-succinimidyl3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble-long-chainN-succinimidyl 3-(2-pyridyldithio) propionate (sulfo-LC-sPDP),succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT),sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamido]hexanoate(sulfo-LC-sMPT),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC),sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBs),m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBs),N-succinimidyl(4-iodoacteyl)aminobenzoate (sIAB),sulfosuccinimidyl(4-iodoacteyl)aminobenzoate (sulfo-sIAB),succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB),sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB),N-(γ-maleimidobutyryloxy)succinimide ester (GMBs),N-(γ-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMBs),succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (sIAXX), succinimidyl4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC),succinimidyl6-((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino) hexanoate(sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl-reactive andsulfhydryl-reactive cross-linkers such as 4-(4-N-maleimidophenyl)butyricacid hydrazide (MPBH),4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8 (M₂C₂H),3-(2-pyridyldithio)propionyl hydrazide (PDPH), amine-reactive andphotoreactive cross-linkers such asN-hydroxysuccinimidyl-4-azidosalicylic acid (NHs-AsA),N-hydroxysulfosuccinimidyl-4-azidosalicylic acid (sulfo-NHs-AsA),sulfosuccinimidyl-(4-azidosalicylamido)hexanoate (sulfo-NHs-LC-AsA),sulfosuccinimidyl-2-(ρ-azidosalicylamido)ethyl-1,3′-dithiopropionate(sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB),N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB),N-succinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate (sANPAH),sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate(sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOs),sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3′-dithiopropionate(sAND), N-succinimidyl-4(4-azidophenyl)1,3′-dithiopropionate (sADP),N-sulfosuccinimidyl(4-azidophenyl)-1,3′-dithiopropionate (sulfo-sADP),sulfosuccinimidyl 4-(ρ-azidophenyl)butyrate (sulfo-sAPB),sulfosuccinimidyl2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3′-dithiopropionate(sAED), sulfosuccinimidyl 7-azido-4-methylcoumain-3-acetate(sulfo-sAMCA), ρ-nitrophenyl diazopyruvate (ρNPDP),ρ-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP),sulfhydryl-reactive and photoreactive cross-linkers such as1-(ρ-Azidosalicylamido)-4-(iodoacetamido)butane (AsIB),N-[4-(ρ-azidosalicylamido)butyl]-3′-(2′-pyridyldithio)propionamide(APDP), benzophenone-4-iodoacetamide, benzophenone-4-maleimidecarbonyl-reactive and photoreactive cross-linkers such as ρ-azidobenzoylhydrazide (ABH), carboxylate-reactive and photoreactive cross-linkerssuch as 4-(ρ-azidosalicylamido)butylamine (AsBA), and arginine-reactiveand photoreactive cross-linkers such as ρ-azidophenyl glyoxal (APG).

In some instances, the linker comprises a reactive functional group. Insome cases, the reactive functional group comprises a nucleophilic groupthat is reactive to an electrophilic group present on a binding moiety.Exemplary electrophilic groups include carbonyl groups—such as aldehyde,ketone, carboxylic acid, ester, amide, enone, acyl halide or acidanhydride. In some embodiments, the reactive functional group isaldehyde. Exemplary nucleophilic groups include hydrazide, oxime, amino,hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.

In some embodiments, the linker comprises a maleimide group. In someinstances, the maleimide group is also referred to as a maleimidespacer. In some instances, the maleimide group further encompasses acaproic acid, forming maleimidocaproyl (mc). In some cases, the linkercomprises maleimidocaproyl (mc). In some cases, the linker ismaleimidocaproyl (mc). In other instances, the maleimide group comprisesa maleimidomethyl group, such assuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC) orsulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-sMCC) described above.

In some embodiments, the maleimide group is a self-stabilizingmaleimide. In some instances, the self-stabilizing maleimide utilizesdiaminopropionic acid (DPR) to incorporate a basic amino group adjacentto the maleimide to provide intramolecular catalysis of tiosuccinimidering hydrolysis, thereby eliminating maleimide from undergoing anelimination reaction through a retro-Michael reaction. In someinstances, the self-stabilizing maleimide is a maleimide group describedin Lyon, et al., “Self-hydrolyzing maleimides improve the stability andpharmacological properties of antibody-drug conjugates,” Nat.Biotechnol. 32(10):1059-1062 (2014). In some instances, the linkercomprises a self-stabilizing maleimide. In some instances, the linker isa self-stabilizing maleimide.

In some embodiments, the linker comprises a peptide moiety. In someinstances, the peptide moiety comprises at least 2, 3, 4, 5, 6, 7, 8, ormore aminoa cid residues. In some instances, the peptide moiety is acleavable peptide moiety (e.g., either enzymatically or chemically). Insome instances, the peptide moiety is a non-cleavable peptide moiety. Insome instances, the peptide moiety comprises Val-Cit(valine-citrulline), Gly-Gly-Phe-Gly (SEQ ID NO: 2111), Phe-Lys,Val-Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg,Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Ala-Leu-Ala-Leu (SEQ ID NO: 2112),or Gly-Phe-Leu-Gly (SEQ ID NO: 2113). In some instances, the linkercomprises a peptide moiety such as: Val-Cit (valine-citrulline),Gly-Gly-Phe-Gly (SEQ ID NO: 2111), Phe-Lys, Val-Lys, Gly-Phe-Lys,Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit,Trp-Cit, Phe-Ala, Ala-Leu-Ala-Leu (SEQ ID NO: 2112), or Gly-Phe-Leu-Gly(SEQ ID NO: 2113). In some cases, the linker comprises Val-Cit. In somecases, the linker is Val-Cit.

In some embodiments, the linker comprises a benzoic acid group, or itsderivatives thereof. In some instances, the benzoic acid group or itsderivatives thereof comprise paraaminobenzoic acid (PABA). In someinstances, the benzoic acid group or its derivatives thereof comprisegamma-aminobutyric acid (GABA).

In some embodiments, the linker comprises one or more of a maleimidegroup, a peptide moiety, and/or a benzoic acid group, in anycombination. In some embodiments, the linker comprises a combination ofa maleimide group, a peptide moiety, and/or a benzoic acid group. Insome instances, the maleimide group is maleimidocaproyl (mc). In someinstances, the peptide group is val-cit. In some instances, the benzoicacid group is PABA. In some instances, the linker comprises a mc-val-citgroup. In some cases, the linker comprises a val-cit-PABA group. Inadditional cases, the linker comprises a mc-val-cit-PABA group.

In some embodiments, the linker is a self-immolative linker or aself-elimination linker. In some cases, the linker is a self-immolativelinker. In other cases, the linker is a self-elimination linker (e.g., acyclization self-elimination linker). In some instances, the linkercomprises a linker described in U.S. Pat. No. 9,089,614 or PCTPublication No. WO2015038426.

In some embodiments, the linker is a dendritic type linker. In someinstances, the dendritic type linker comprises a branching,multifunctional linker moiety. In some instances, the dendritic typelinker is used to increase the molar ratio of polynucleotide B to thebinding moiety A. In some instances, the dendritic type linker comprisesPAMAM dendrimers.

In some embodiments, the linker is a traceless linker or a linker inwhich after cleavage does not leave behind a linker moiety (e.g., anatom or a linker group) to a binding moiety A, a polynucleotide B, apolymer C, or an endosomolytic moiety D. Exemplary traceless linkersinclude, but are not limited to, germanium linkers, silicium linkers,sulfur linkers, selenium linkers, nitrogen linkers, phosphorus linkers,boron linkers, chromium linkers, or phenylhydrazide linker. In somecases, the linker is a traceless aryl-triazene linker as described inHejesen, et al., “A traceless aryl-triazene linker for DNA-directedchemistry,” Org Biomol Chem 11(15): 2493-2497 (2013). In some instances,the linker is a traceless linker described in Blaney, et al., “Tracelesssolid-phase organic synthesis,” Chem. Rev. 102: 2607-2024 (2002). Insome instances, a linker is a traceless linker as described in U.S. Pat.No. 6,821,783.

In some instances, the linker comprises a functional group that exertssteric hinderance at the site of bonding between the linker and aconjugating moiety (e.g., A, B, C, or D described herein). In someinstances, the steric hinderance is a steric hindrance around adisulfide bond. Exemplary linkers that exhibit steric hinderancecomprises a heterobifuctional linker, such as a heterobifuctional linkerdescribed above. In some cases, a linker that exhibits steric hinderancecomprises SMCC and SPDB.

In some instances, the linker is an acid cleavable linker. In someinstances, the acid cleavable linker comprises a hydrazone linkage,which is susceptible to hydrolytic cleavage. In some cases, the acidcleavable linker comprises a thiomaleamic acid linker. In some cases,the acid cleavable linker is a thiomaleamic acid linker as described inCastaneda, et al, “Acid-cleavable thiomaleamic acid linker forhomogeneous antibody-drug conjugation,” Chem. Commun. 49: 8187-8189(2013).

In some instances, the linker is a linker described in U.S. Pat. Nos.6,884,869; 7,498,298; 8,288,352; 8,609,105; or 8,697,688; U.S. PatentPublication Nos. 2014/0127239; 2013/028919; 2014/286970; 2013/0309256;2015/037360; or 2014/0294851; or PCT Publication Nos. WO2015057699;WO2014080251; WO2014197854; WO2014145090; or WO2014177042.

In some embodiments, X, Y, and L are independently a bond or a linker.In some instances, X, Y, and L are independently a bond. In some cases,X, Y, and L are independently a linker.

In some instances, X is a bond or a linker. In some instances, X is abond. In some instances, X is a linker. In some instances, the linker isa C₁-C₆ alkyl group. In some cases, X is a C₁-C₆ alkyl group, such asfor example, a C₅, C₄, C₃, C₂, or C₁ alkyl group. In some cases, theC₁-C₆ alkyl group is an unsubstituted C₁-C₆ alkyl group. As used in thecontext of a linker, and in particular in the context of X, alkyl meansa saturated straight or branched hydrocarbon radical containing up tosix carbon atoms. In some instances, X is a non-polymeric linker. Insome instances, X includes a homobifuctional linker or aheterobifuctional linker described supra. In some cases, X includes aheterobifunctional linker. In some cases, X includes sMCC. In otherinstances, X includes a heterobifuctional linker optionally conjugatedto a C₁-C₆ alkyl group. In other instances, X includes sMCC optionallyconjugated to a C₁-C₆ alkyl group. In additional instances, X does notinclude a homobifuctional linker or a heterobifunctional linkerdescribed supra.

In some instances, Y is a bond or a linker. In some instances, Y is abond. In other cases, Y is a linker. In some embodiments, Y is a C₁-C₆alkyl group. In some instances, Y is a homobifuctional linker or aheterobifunctional linker described supra. In some instances, Y is ahomobifuctional linker described supra. In some instances, Y is aheterobifunctional linker described supra. In some instances, Ycomprises a maleimide group, such as maleimidocaproyl (mc) or aself-stabilizing maleimide group described above. In some instances, Ycomprises a peptide moiety, such as Val-Cit. In some instances, Ycomprises a benzoic acid group, such as PABA. In additional instances, Ycomprises a combination of a maleimide group, a peptide moiety, and/or abenzoic acid group. In additional instances, Y comprises a mc group. Inadditional instances, Y comprises a mc-val-cit group. In additionalinstances, Y comprises a val-cit-PABA group. In additional instances, Ycomprises a mc-val-cit-PABA group.

In some instances, L is a bond or a linker. In some cases, L is a bond.In other cases, L is a linker. In some embodiments, L is a C₁-C₆ alkylgroup. In some instances, L is a homobifuctional linker or aheterobifunctional linker described supra. In some instances, L is ahomobifuctional linker described supra. In some instances, L is aheterobifunctional linker described supra. In some instances, Lcomprises a maleimide group, such as maleimidocaproyl (mc) or aself-stabilizing maleimide group described above. In some instances, Lcomprises a peptide moiety, such as Val-Cit. In some instances, Lcomprises a benzoic acid group, such as PABA. In additional instances, Lcomprises a combination of a maleimide group, a peptide moiety, and/or abenzoic acid group. In additional instances, L comprises a mc group. Inadditional instances, L comprises a mc-val-cit group. In additionalinstances, L comprises a val-cit-PABA group. In additional instances, Lcomprises a mc-val-cit-PABA group.

Methods of Use

In some embodiments, a composition or a pharmaceutical formulationdescribed herein comprising a binding moiety conjugated to a polynucleicacid molecule and a polymer is used for the treatment of a disease ordisorder. In some instances, the disease or disorder is a cancer. Insome embodiments, a composition or a pharmaceutical formulationdescribed herein is used as an immunotherapy for the treatment of adisease or disorder. In some instances, the immunotherapy is animmuno-oncology therapy.

Cancer

In some embodiments, a composition or a pharmaceutical formulationdescribed herein is used for the treatment of cancer. In some instances,the cancer is a solid tumor. In some instances, the cancer is ahematologic malignancy. In some instances, the cancer is a relapsed orrefractory cancer, or a metastatic cancer. In some instances, the solidtumor is a relapsed or refractory solid tumor, or a metastatic solidtumor. In some cases, the hematologic malignancy is a relapsed orrefractory hematologic malignancy, or a metastatic hematologicmalignancy.

In some embodiments, the cancer is a solid tumor. Exemplary solid tumorincludes, but is not limited to, anal cancer, appendix cancer, bile ductcancer (i.e., cholangiocarcinoma), bladder cancer, brain tumor, breastcancer, cervical cancer, colon cancer, cancer of Unknown Primary (CUP),esophageal cancer, eye cancer, fallopian tube cancer,gastroenterological cancer, kidney cancer, liver cancer, lung cancer,medulloblastoma, melanoma, oral cancer, ovarian cancer, pancreaticcancer, parathyroid disease, penile cancer, pituitary tumor, prostatecancer, rectal cancer, skin cancer, stomach cancer, testicular cancer,throat cancer, thyroid cancer, uterine cancer, vaginal cancer, or vulvarcancer.

In some instances, a composition or a pharmaceutical formulationdescribed herein comprising a binding moiety conjugated to a polynucleicacid molecule and a polymer is used for the treatment of a solid tumor.In some instances, a composition or a pharmaceutical formulationdescribed herein comprising a binding moiety conjugated to a polynucleicacid molecule and a polymer is used for the treatment of anal cancer,appendix cancer, bile duct cancer (i.e., cholangiocarcinoma), bladdercancer, brain tumor, breast cancer, cervical cancer, colon cancer,cancer of Unknown Primary (CUP), esophageal cancer, eye cancer,fallopian tube cancer, gastroenterological cancer, kidney cancer, livercancer, lung cancer, medulloblastoma, melanoma, oral cancer, ovariancancer, pancreatic cancer, parathyroid disease, penile cancer, pituitarytumor, prostate cancer, rectal cancer, skin cancer, stomach cancer,testicular cancer, throat cancer, thyroid cancer, uterine cancer,vaginal cancer, or vulvar cancer. In some instances, the solid tumor isa relapsed or refractory solid tumor, or a metastatic solid tumor.

In some instances, the cancer is a hematologic malignancy. In someinstances, the hematologic malignancy is a leukemia, a lymphoma, amyeloma, a non-Hodgkin's lymphoma, or a Hodgkin's lymphoma. In someinstances, the hematologic malignancy comprises chronic lymphocyticleukemia (CLL), small lymphocytic lymphoma (SLL), high risk CLL, anon-CLL/SLL lymphoma, prolymphocytic leukemia (PLL), follicular lymphoma(FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL),Waldenström's macroglobulinemia, multiple myeloma, extranodal marginalzone B cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt'slymphoma, non-Burkitt high grade B cell lymphoma, primary mediastinalB-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursorB-lymphoblastic lymphoma, B cell prolymphocytic leukemia,lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cellmyeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma,intravascular large B cell lymphoma, primary effusion lymphoma, orlymphomatoid granulomatosis.

In some instances, a composition or a pharmaceutical formulationdescribed herein comprising a binding moiety conjugated to a polynucleicacid molecule and a polymer is used for the treatment of a hematologicmalignancy. In some instances, a composition or a pharmaceuticalformulation described herein comprising a binding moiety conjugated to apolynucleic acid molecule and a polymer is used for the treatment of aleukemia, a lymphoma, a myeloma, a non-Hodgkin's lymphoma, or aHodgkin's lymphoma. In some instances, the hematologic malignancycomprises chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma(SLL), high risk CLL, a non-CLL/SLL lymphoma, prolymphocytic leukemia(PLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL),mantle cell lymphoma (MCL), Waldenström's macroglobulinemia, multiplemyeloma, extranodal marginal zone B cell lymphoma, nodal marginal zone Bcell lymphoma, Burkitt's lymphoma, non-Burkitt high grade B celllymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblasticlarge cell lymphoma, precursor B-lymphoblastic lymphoma, B cellprolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginalzone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic)large B cell lymphoma, intravascular large B cell lymphoma, primaryeffusion lymphoma, or lymphomatoid granulomatosis. In some cases, thehematologic malignancy is a relapsed or refractory hematologicmalignancy, or a metastatic hematologic malignancy.

In some instances, the cancer is a KRAS-associated, EGFR-associated,AR-associated cancer, HPRT1-associated cancer, or β-catenin associatedcancer. In some instances, a composition or a pharmaceutical formulationdescribed herein comprising a binding moiety conjugated to a polynucleicacid molecule and a polymer is used for the treatment of aKRAS-associated, EGFR-associated, AR-associated cancer, HPRT1-associatedcancer, or β-catenin associated cancer. In some instances, a compositionor a pharmaceutical formulation described herein comprising a bindingmoiety conjugated to a polynucleic acid molecule and a polymer is usedfor the treatment of a KRAS-associated cancer. In some instances, acomposition or a pharmaceutical formulation described herein comprisinga binding moiety conjugated to a polynucleic acid molecule and a polymeris used for the treatment of an EGFR-associated cancer. In someinstances, a composition or a pharmaceutical formulation describedherein comprising a binding moiety conjugated to a polynucleic acidmolecule and a polymer is used for the treatment of an AR-associatedcancer. In some instances, a composition or a pharmaceutical formulationdescribed herein comprising a binding moiety conjugated to a polynucleicacid molecule and a polymer is used for the treatment of anHPRT1-associated cancer. In some instances, a composition or apharmaceutical formulation described herein comprising a binding moietyconjugated to a polynucleic acid molecule and a polymer is used for thetreatment of a β-catenin associated cancer. In some instances, thecancer is a solid tumor. In some instances, the cancer is a hematologicmalignancy. In some instances, the solid tumor is a relapsed orrefractory solid tumor, or a metastatic solid tumor. In some cases, thehematologic malignancy is a relapsed or refractory hematologicmalignancy, or a metastatic hematologic malignancy. In some instances,the cancer comprises bladder cancer, breast cancer, colorectal cancer,endometrial cancer, esophageal cancer, glioblastoma multiforme, head andneck cancer, kidney cancer, lung cancer, ovarian cancer, pancreaticcancer, prostate cancer, thyroid cancer, acute myeloid leukemia, CLL,DLBCL, or multiple myeloma. In some instances, the β-catenin associatedcancer further comprises PIK3C-associated cancer and/or MYC-associatedcancer.

Immunotherapy

In some embodiments, a composition or a pharmaceutical formulationdescribed herein is used as an immunotherapy for the treatment of adisease or disorder. In some instances, the immunotherapy is animmuno-oncology therapy. In some instances, immuno-oncology therapy iscategorized into active, passive, or combinatory (active and passive)methods. In active immuno-oncology therapy method, for example,tumor-associated antigens (TAAs) are presented to the immune system totrigger an attack on cancer cells presenting these TAAs. In someinstances, the active immune-oncology therapy method includestumor-targeting and/or immune-targeting agents (e.g., checkpointinhibitor agents such as monoclonal antibodies), and/or vaccines, suchas in situ vaccination and/or cell-based or non-cell based (e.g.,dendritic cell-based, tumor cell-based, antigen, anti-idiotype, DNA, orvector-based) vaccines. In some instances, the cell-based vaccines arevaccines which are generated using activated immune cells obtained froma patient's own immune system which are then activated by the patient'sown cancer. In some instances, the active immune-oncology therapy isfurther subdivided into non-specific active immunotherapy and specificactive immunotherapy. In some instances, non-specific activeimmunotherapy utilizes cytokines and/or other cell signaling componentsto induce a general immune system response. In some cases, specificactive immunotherapy utilizes specific TAAs to elicite an immuneresponse.

In some embodiments, a composition or a pharmaceutical formulationdescribed herein is used as an active immuno-oncology therapy method forthe treatment of a disease or disorder (e.g., cancer). In someembodiments, the composition or a pharmaceutical formulation describedherein comprises a tumor-targeting agent. In some instances, thetumor-targeting agent is encompassed by a binding moiety A. In otherinstances, the tumor-targeting agent is an additional agent used incombination with a molecule of Formula (I). In some instances, thetumor-targeting agent is a tumor-directed polypeptide (e.g., atumor-directed antibody). In some instances, the tumor-targeting agentis a tumor-directed antibody, which exerts its antitumor activitythrough mechanisms such as direct killing (e.g., signaling-inducedapoptosis), complement-dependent cytotoxicity (CDC), and/orantibody-dependent cell-mediated cytotoxicity (ADCC). In additionalinstances, the tumor-targeting agent elicits an adaptive immuneresponse, with the induction of antitumor T cells.

In some embodiments, the binding moiety A is a tumor-directedpolypeptide (e.g., a tumor-directed antibody). In some instances, thebinding moiety A is a tumor-directed antibody, which exerts itsantitumor activity through mechanisms such as direct killing (e.g.,signaling-induced apoptosis), complement-dependent cytotoxicity (CDC),and/or antibody-dependent cell-mediated cytotoxicity (ADCC). Inadditional instances, the binding moiety A elicits an adaptive immuneresponse, with the induction of antitumor T cells.

In some embodiments, the composition or a pharmaceutical formulationdescribed herein comprises an immune-targeting agent. In some instances,the immune-targeting agent is encompassed by a binding moiety A. Inother instances, the immune-targeting agent is an additional agent usedin combination with a molecule of Formula (I). In some instances, theimmune-targeting agent comprises cytokines, checkpoint inhibitors, or acombination thereof.

In some embodiments, the immune-targeting agent is a checkpointinhibitor. In some cases, an immune checkpoint molecule is a moleculepresented on the cell surface of CD4 and/or CD8 T cells. Exemplaryimmune checkpoint molecules include, but are not limited to, ProgrammedDeath-Ligand 1 (PD-L1, also known as B7-H1, CD274), Programmed Death 1(PD-1), CTLA-4, B7H1, B7H4, OX-40, CD137, CD40, 2B4, IDO1, IDO2, VISTA,CD27, CD28, PD-L2 (B7-DC, CD273), LAG3, CD80, CD86, PDL2, B7H3, HVEM,BTLA, KIR, GAL9, TIM3, A2aR, MARCO (macrophage receptor withcollageneous structure), PS (phosphatidylserine), ICOS (inducible T cellcostimulator), HAVCR2, CD276, VTCN1, CD70, and CD160.

In some instances, an immune checkpoint inhibitor refers to any moleculethat modulates or inhibits the activity of an immune checkpointmolecule. In some instances, immune checkpoint inhibitors includeantibodies, antibody-derivatives (e.g., Fab fragments, scFvs,minobodies, diabodies), antisense oligonucleotides, siRNA, aptamers, orpeptides. In some embodiments, an immune checkpoint inhibitor is aninhibitor of Programmed Death-Ligand 1 (PD-L1, also known as B7-H1,CD274), Programmed Death 1 (PD-1), CTLA-4, PD-L2 (B7-DC, CD273), LAG3,TIM3, 2B4, A2aR, B7H1, B7H3, B7H4, BTLA, CD2, CD27, CD28, CD30, CD40,CD70, CD80, CD86, CD137, CD160, CD226, CD276, DR3, GAL9, GITR, HAVCR2,HVEM, IDO1, IDO2, ICOS (inducible T cell costimulator), KIR, LAIR1,LIGHT, MARCO (macrophage receptor with collageneous structure), PS(phosphatidylserine), OX-40, SLAM, TIGHT, VISTA, VTCN1, or anycombinations thereof.

In some embodiments, exemplary checkpoint inhibitors include:

PD-L1 inhibitors such as Genentech's MPDL3280A (RG7446), Anti-mousePD-L1 antibody Clone 10F.9G2 (Cat # BE0101) from BioXcell, anti-PD-L1monoclonal antibody MDX-1105 (BMS-936559) and BMS-935559 fromBristol-Meyer's Squibb, MSB0010718C, mouse anti-PD-L1 Clone 29E.2A3, andAstraZeneca's MEDI4736;

PD-L2 inhibitors such as GlaxoSmithKline's AMP-224 (Amplimmune), andrHIgM12B7;

PD-1 inhibitors such as anti-mouse PD-1 antibody Clone J43 (Cat #BE0033-2) from BioXcell, anti-mouse PD-1 antibody Clone RMP1-14 (Cat #BE0146) from BioXcell, mouse anti-PD-1 antibody Clone EH12, Merck'sMK-3475 anti-mouse PD-1 antibody (Keytruda, pembrolizumab,lambrolizumab), AnaptysBio's anti-PD-1 antibody known as ANB011,antibody MDX-1 106 (ONO-4538), Bristol-Myers Squibb's human IgG4monoclonal antibody nivolumab (Opdivo®, BMS-936558, MDX1106),AstraZeneca's AMP-514 and AMP-224, and Pidilizumab (CT-011) fromCureTech Ltd;

CTLA-4 inhibitors such as Bristol Meyers Squibb's anti-CTLA-4 antibodyipilimumab (also known as Yervoy®, MDX-010, BMS-734016 and MDX-101),anti-CTLA4 Antibody, clone 9H10 from Millipore, Pfizer's tremelimumab(CP-675,206, ticilimumab), and anti-CTLA4 antibody clone BNI3 fromAbcam;

LAG3 inhibitors such as anti-Lag-3 antibody clone eBioC9B7W (C9B7W) fromeBioscience, anti-Lag3 antibody LS-B2237 from LifeSpan Biosciences,IMP321 (ImmuFact) from Immutep, anti-Lag3 antibody BMS-986016, and theLAG-3 chimeric antibody A9H12;

B7-H3 inhibitors such as MGA271;

KIR inhibitors such as Lirilumab (IPH2101);

CD137 (41BB) inhibitors such as urelumab (BMS-663513, Bristol-MyersSquibb), PF-05082566 (anti-4-1BB, PF-2566, Pfizer), or XmAb-5592(Xencor);

PS inhibitors such as Bavituximab;

and inhibitors such as an antibody or fragments (e.g., a monoclonalantibody, a human, humanized, or chimeric antibody) thereof, RNAimolecules, or small molecules to TIM3, CD52, CD30, CD20, CD33, CD27,OX40 (CD134), GITR, ICOS, BTLA (CD272), CD160, 2B4, LAIR1, TIGHT, LIGHT,DR3, CD226, CD2, or SLAM.

In some embodiments, a binding moiety A comprising an immune checkpointinhibitor is used for the treatment of a disease or disorder (e.g.,cancer). In some instances, the binding moiety A is a bispecificantibody or a binding fragment thereof that comprises an immunecheckpoint inhibitor. In some cases, a binding moiety A comprising aninhibitor of Programmed Death-Ligand 1 (PD-L1, also known as B7-HI,CD274), Programmed Death 1 (PD-1), CTLA-4, PD-L2 (B7-DC, CD273), LAG3,TIM3, 2B4, A2aR, B7H1, B7H3, B7H4, BTLA, CD2, CD27, CD28, CD30, CD40,CD70, CD80, CD86, CD137, CD160, CD226, CD276, DR3, GAL9, GITR, HAVCR2,HVEM, IDO1, IDO2, ICOS (inducible T cell costimulator), KIR, LAIR1,LIGHT, MARCO (macrophage receptor with collageneous structure), PS(phosphatidylserine), OX-40, SLAM, TIGHT, VISTA, VTCN1, or anycombinations thereof, is used for the treatment of a disease or disorder(e.g., cancer).

In some embodiments, a molecule of Formula (I) in combination with animmune checkpoint inhibitor is used for the treatment of a disease ordisorder (e.g., cancer). In some instances, the immune checkpointinhibitor comprises an inhibitor of Programmed Death-Ligand 1 (PD-L1,also known as B7-HI, CD274), Programmed Death 1 (PD-1), CTLA-4, PD-L2(B7-DC, CD273), LAG3, TIM3, 2B4, A2aR, B7H1, B7H3, B7H4, BTLA, CD2,CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137, CD160, CD226, CD276,DR3, GAL9, GITR, HAVCR2, HVEM, IDO1, IDO2, ICOS (inducible T cellcostimulator), KIR, LAIR1, LIGHT, MARCO (macrophage receptor withcollageneous structure), PS (phosphatidylserine), OX-40, SLAM, TIGHT,VISTA, VTCN1, or any combinations thereof. In some cases, a molecule ofFormula (I) is used in combination with ipilimumab, tremelimumab,nivolumab, pemrolizumab, pidilizumab, MPDL3280A, MEDI4736, MSB0010718C,MK-3475, or BMS-936559, for the treatment of a disease or disorder(e.g., cancer).

In some embodiments, the immune-targeting agent is a cytokine. In somecases, cytokine is further subgrouped into chemokine, interferon,interleukin, and tumor necrosis factor. In some embodiments, chemokineplays a role as a chemoattractant to guide the migration of cells, andis classified into four subfamilies: CXC, CC, CX3C, and XC. Exemplarychemokines include chemokines from the CC subfamily: CCL1, CCL2 (MCP-1),CCL3, CCL4, CCL5 (RANTES), CCL6, CCL7, CCL8, CCL9 (or CCL10), CCL11,CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21,CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, and CCL28; the CXC subfamily:CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10,CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, and CXCL17; the XCsubfamily: XCL1 and XCL2; and the CX3C subfamily CX3CL1.

Interferon (IFNs) comprises interferon type I (e.g. IFN-α, IFN-β, IFN-ε,IFN-κ, and IFN-ω), interferon type II (e.g. IFN-γ), and interferon typeIII. In some embodiments, IFN-α is further classified into about 13subtypes which include IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8,IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, and IFNA21.

Interleukin is expressed by leukocyte or white blood cell and promotethe development and differentiation of T and B lymphocytes andhematopoietic cells. Exemplary interleukins include IL-1, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9, IL-10, IL-11, IL-12, IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23,IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33,IL-35, and IL-36.

Tumor necrosis factors (TNFs) are a group of cytokines that modulateapoptosis. In some instances, there are about 19 members within the TNFfamily, including, not limited to, TNFα, lymphotoxin-alpha (LT-alpha),lymphotoxin-beta (LT-beta), T cell antigen gp39 (CD40L), CD27L, CD30L,FASL, 4-IBBL, OX40L, and TNF-related apoptosis inducing ligand (TRAIL).

In some embodiments, a molecule of Formula (I) in combination with acytokine is used for the treatment of a disease or disorder (e.g.,cancer). In some cases, a molecule of Formula (I) in combination with achemokine is used for the treatment of a disease or disorder (e.g.,cancer). In some cases, a molecule of Formula (I) in combination with aninterferon is used for the treatment of a disease or disorder (e.g.,cancer). In some cases, a molecule of Formula (I) in combination with aninterleukin is used for the treatment of a disease or disorder (e.g.,cancer). In some cases, a molecule of Formula (I) in combination with atumor necrosis factor is used for the treatment of a disease or disorder(e.g., cancer). In some instances, a molecule of Formula (I) incombination with IL-113, IL-2, IL-7, IL-8, IL-15, MCP-1 (CCL2), MIP-1α,RANTES, MCP-3, MIP5, CCL19, CCL21, CXCL2, CXCL9, CXCL10, or CXCL11 isused for the treatment of a disease or disorder (e.g., cancer).

In some embodiments, the composition or a pharmaceutical formulationdescribed herein comprises a vaccine. In some instances, the vaccine isan in situ vaccination. In some instances, the vaccine is a cell-basedvaccine. In some instances, the vaccine is a non-cell based vaccine. Insome instances, a molecule of Formula (I) in combination with dendriticcell-based vaccine is used for the treatment of a disease or disorder(e.g., cancer). In some instances, a molecule of Formula (I) incombination with tumor cell-based vaccine is used for the treatment of adisease or disorder (e.g., cancer). In some instances, a molecule ofFormula (I) in combination with antigen vaccine is used for thetreatment of a disease or disorder (e.g., cancer). In some instances, amolecule of Formula (I) in combination with anti-idiotype vaccine isused for the treatment of a disease or disorder (e.g., cancer). In someinstances, a molecule of Formula (I) in combination with DNA vaccine isused for the treatment of a disease or disorder (e.g., cancer). In someinstances, a molecule of Formula (I) in combination with vector-basedvaccine is used for the treatment of a disease or disorder (e.g.,cancer).

In some embodiments, a composition or a pharmaceutical formulationdescribed herein is used as a passive immuno-oncology therapy method forthe treatment of a disease or disorder (e.g., cancer). The passivemethod, in some instances, utilizes adoptive immune system componentssuch as T cells, natural killer (NK) T cells, and/or chimeric antigenreceptor (CAR) T cells generated exogenously to attack cancer cells.

In some embodiments, a molecule of Formula (I) in combination with aT-cell based therapeutic agent is used for the treatment of a disease ordisorder (e.g., cancer). In some cases, the T-cell based therapeuticagent is an activated T-cell agent that recognizes one or more of a CDcell surface marker described above. In some instances, the T-cell basedtherapeutic agent comprises an activated T-cell agent that recognizesone or more of CD2, CD3, CD4, CD5, CD8, CD27, CD28, CD80, CD134, CD137,CD152, CD154, CD160, CD200R, CD223, CD226, CD244, CD258, CD267, CD272,CD274, CD278, CD279, or CD357. In some instances, a molecule of Formula(I) in combination with an activated T-cell agent recognizing one ormore of CD2, CD3, CD4, CD5, CD8, CD27, CD28, CD80, CD134, CD137, CD152,CD154, CD160, CD200R, CD223, CD226, CD244, CD258, CD267, CD272, CD274,CD278, CD279, or CD357 is used for the treatment of a disease ordisorder (e.g., cancer).

In some embodiments, a molecule of Formula (I) in combination withnatural killer (NK) T cell-based therapeutic agent is used for thetreatment of a disease or disorder (e.g., cancer). In some instances,the NK-based therapeutic agent is an activated NK agent that recognizesone or more of a CD cell surface marker described above. In some cases,the NK-based therapeutic agent is an activated NK agent that recognizesone or more of CD2, CD11a, CD11b, CD16, CD56, CD58, CD62L, CD85j,CD158a/b, CD158c, CD158e/f/k, CD158h/j, CD159a, CD162, CD226, CD314,CD335, CD337, CD244, or CD319. In some instances, a molecule of Formula(I) in combination with an activated NK agent recognizing one or more ofCD2, CD11a, CD11b, CD16, CD56, CD58, CD62L, CD85j, CD158a/b, CD158c,CD158e/f/k, CD158h/j, CD159a, CD162, CD226, CD314, CD335, CD337, CD244,or CD319 is used for the treatment of a disease or disorder (e.g.,cancer).

In some embodiments, a molecule of Formula (I) in combination with CAR-Tcell-based therapeutic agent is used for the treatment of a disease ordisorder (e.g., cancer).

In some embodiments, a molecule of Formula (I) in combination with anadditional agent that destabilizes the endosomal membrane (or disruptsthe endosomal-lysosomal membrane trafficking) is used for the treatmentof a disease or disorder (e.g., cancer). In some instances, theadditional agent comprises an antimitotic agent. Exemplary antimitoticagents include, but are not limited to, taxanes such as paclitaxel anddocetaxel; vinca alkaloids such as vinblastine, vincristine, vindesine,and vinorelbine; cabazitaxel; colchicine; eribulin; estramustine;etoposide; ixabepilone; podophyllotoxin; teniposide; or griseofulvin. Insome instances, the additional agent comprises paclitaxel, docetaxel,vinblastine, vincristine, vindesine, vinorelbine, cabazitaxel,colchicine, eribulin, estramustine, etoposide, ixabepilone,podophyllotoxin, teniposide, or griseofulvin. In some instances, theadditional agent comprises taxol. In some instances, the additionalagent comprises paclitaxel. In some instances, the additional agentcomprises etoposide. In other instances, the additional agent comprisesvitamin K3.

In some embodiments, a composition or a pharmaceutical formulationdescribed herein is used as a combinatory method (including for bothactive and passive methods) in the treatment of a disease or disorder(e.g., cancer).

Pharmaceutical Formulation

In some embodiments, the pharmaceutical formulations described hereinare administered to a subject by multiple administration routes,including but not limited to, parenteral (e.g., intravenous,subcutaneous, intramuscular), oral, intranasal, buccal, rectal, ortransdermal administration routes. In some instances, the pharmaceuticalcomposition describe herein is formulated for parenteral (e.g.,intravenous, subcutaneous, intramuscular) administration. In otherinstances, the pharmaceutical composition describe herein is formulatedfor oral administration. In still other instances, the pharmaceuticalcomposition describe herein is formulated for intranasal administration.

In some embodiments, the pharmaceutical formulations include, but arenot limited to, aqueous liquid dispersions, self-emulsifyingdispersions, solid solutions, liposomal dispersions, aerosols, soliddosage forms, powders, immediate-release formulations,controlled-release formulations, fast melt formulations, tablets,capsules, pills, delayed release formulations, extended releaseformulations, pulsatile release formulations, multiparticulateformulations (e.g., nanoparticle formulations), and mixed immediate andcontrolled release formulations.

In some instances, the pharmaceutical formulation includesmultiparticulate formulations. In some instances, the pharmaceuticalformulation includes nanoparticle formulations. In some instances,nanoparticles comprise cMAP, cyclodextrin, or lipids. In some cases,nanoparticles comprise solid lipid nanoparticles, polymericnanoparticles, self-emulsifying nanoparticles, liposomes,microemulsions, or micellar solutions. Additional exemplarynanoparticles include, but are not limited to, paramagneticnanoparticles, superparamagnetic nanoparticles, metal nanoparticles,fullerene-like materials, inorganic nanotubes, dendrimers (such as withcovalently attached metal chelates), nanofibers, nanohorns, nano-onions,nanorods, nanoropes and quantum dots. In some instances, a nanoparticleis a metal nanoparticle, e.g., a nanoparticle of scandium, titanium,vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc,yttrium, zirconium, niobium, molybdenum, ruthenium, rhodium, palladium,silver, cadmium, hafnium, tantalum, tungsten, rhenium, osmium, iridium,platinum, gold, gadolinium, aluminum, gallium, indium, tin, thallium,lead, bismuth, magnesium, calcium, strontium, barium, lithium, sodium,potassium, boron, silicon, phosphorus, germanium, arsenic, antimony, andcombinations, alloys or oxides thereof.

In some instances, a nanoparticle includes a core or a core and a shell,as in a core-shell nanoparticle.

In some instances, a nanoparticle is further coated with molecules forattachment of functional elements (e.g., with one or more of apolynucleic acid molecule or binding moiety described herein). In someinstances, a coating comprises chondroitin sulfate, dextran sulfate,carboxymethyl dextran, alginic acid, pectin, carragheenan, fucoidan,agaropectin, porphyran, karaya gum, gellan gum, xanthan gum, hyaluronicacids, glucosamine, galactosamine, chitin (or chitosan), polyglutamicacid, polyaspartic acid, lysozyme, cytochrome C, ribonuclease,trypsinogen, chymotrypsinogen, α-chymotrypsin, polylysine, polyarginine,histone, protamine, ovalbumin, dextrin, or cyclodextrin. In someinstances, a nanoparticle comprises a graphene-coated nanoparticle.

In some cases, a nanoparticle has at least one dimension of less thanabout 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm.

In some instances, the nanoparticle formulation comprises paramagneticnanoparticles, superparamagnetic nanoparticles, metal nanoparticles,fullerene-like materials, inorganic nanotubes, dendrimers (such as withcovalently attached metal chelates), nanofibers, nanohorns, nano-onions,nanorods, nanoropes or quantum dots. In some instances, a polynucleicacid molecule or a binding moiety described herein is conjugated eitherdirectly or indirectly to the nanoparticle. In some instances, at least1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more polynucleicacid molecules or binding moieties described herein are conjugatedeither directly or indirectly to a nanoparticle.

In some embodiments, the pharmaceutical formulations include a carrieror carrier materials selected on the basis of compatibility with thecomposition disclosed herein, and the release profile properties of thedesired dosage form. Exemplary carrier materials include, e.g., binders,suspending agents, disintegration agents, filling agents, surfactants,solubilizers, stabilizers, lubricants, wetting agents, diluents, and thelike. Pharmaceutically compatible carrier materials include, but are notlimited to, acacia, gelatin, colloidal silicon dioxide, calciumglycerophosphate, calcium lactate, maltodextrin, glycerine, magnesiumsilicate, polyvinylpyrrollidone (PVP), cholesterol, cholesterol esters,sodium caseinate, soy lecithin, taurocholic acid, phosphotidylcholine,sodium chloride, tricalcium phosphate, dipotassium phosphate, celluloseand cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan,monoglyceride, diglyceride, pregelatinized starch, and the like. See,e.g., Remington: The Science and Practice of Pharmacy, Nineteenth Ed(Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E.,Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical DosageForms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical DosageForms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams &Wilkins 1999).

In some instances, the pharmaceutical formulations further includepH-adjusting agents or buffering agents which include acids such asacetic, boric, citric, lactic, phosphoric and hydrochloric acids; basessuch as sodium hydroxide, sodium phosphate, sodium borate, sodiumcitrate, sodium acetate, sodium lactate andtris-hydroxymethylaminomethane; and buffers such as citrate/dextrose,sodium bicarbonate and ammonium chloride. Such acids, bases and buffersare included in an amount required to maintain pH of the composition inan acceptable range.

In some instances, the pharmaceutical formulation includes one or moresalts in an amount required to bring osmolality of the composition intoan acceptable range. Such salts include those having sodium, potassiumor ammonium cations and chloride, citrate, ascorbate, borate, phosphate,bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable saltsinclude sodium chloride, potassium chloride, sodium thiosulfate, sodiumbisulfite and ammonium sulfate.

In some instances, the pharmaceutical formulations further includediluent which are used to stabilize compounds because they can provide amore stable environment. Salts dissolved in buffered solutions (whichalso can provide pH control or maintenance) are utilized as diluents inthe art, including, but not limited to a phosphate buffered salinesolution. In certain instances, diluents increase bulk of thecomposition to facilitate compression or create sufficient bulk forhomogenous blend for capsule filling. Such compounds can include e.g.,lactose, starch, mannitol, sorbitol, dextrose, microcrystallinecellulose such as Avicel®; dibasic calcium phosphate, dicalciumphosphate dihydrate; tricalcium phosphate, calcium phosphate; anhydrouslactose, spray-dried lactose; pregelatinized starch, compressible sugar,such as Di-Pac® (Amstar); mannitol, hydroxypropylmethylcellulose,hydroxypropylmethylcellulose acetate stearate, sucrose-based diluents,confectioner's sugar; monobasic calcium sulfate monohydrate, calciumsulfate dihydrate; calcium lactate trihydrate, dextrates; hydrolyzedcereal solids, amylose; powdered cellulose, calcium carbonate; glycine,kaolin; mannitol, sodium chloride; inositol, bentonite, and the like.

In some cases, the pharmaceutical formulations include disintegrationagents or disintegrants to facilitate the breakup or disintegration of asubstance. The term “disintegrate” include both the dissolution anddispersion of the dosage form when contacted with gastrointestinalfluid. Examples of disintegration agents include a starch, e.g., anatural starch such as corn starch or potato starch, a pregelatinizedstarch such as National 1551 or Amijel®, or sodium starch glycolate suchas Promogel® or Explotab®, a cellulose such as a wood product,methylcrystalline cellulose, e.g., Avicel®, Avicel® PH101, Avicel®PH102, Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, Ming Tia®, andSolka-Floc®, methylcellulose, croscarmellose, or a cross-linkedcellulose, such as cross-linked sodium carboxymethylcellulose(Ac-Di-Sol®), cross-linked carboxymethylcellulose, or cross-linkedcroscarmellose, a cross-linked starch such as sodium starch glycolate, across-linked polymer such as crospovidone, a cross-linkedpolyvinylpyrrolidone, alginate such as alginic acid or a salt of alginicacid such as sodium alginate, a clay such as Veegum® HV (magnesiumaluminum silicate), a gum such as agar, guar, locust bean, Karaya,pectin, or tragacanth, sodium starch glycolate, bentonite, a naturalsponge, a surfactant, a resin such as a cation-exchange resin, citruspulp, sodium lauryl sulfate, sodium lauryl sulfate in combinationstarch, and the like.

In some instances, the pharmaceutical formulations include fillingagents such as lactose, calcium carbonate, calcium phosphate, dibasiccalcium phosphate, calcium sulfate, microcrystalline cellulose,cellulose powder, dextrose, dextrates, dextran, starches, pregelatinizedstarch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride,polyethylene glycol, and the like.

Lubricants and glidants are also optionally included in thepharmaceutical formulations described herein for preventing, reducing orinhibiting adhesion or friction of materials. Exemplary lubricantsinclude, e.g., stearic acid, calcium hydroxide, talc, sodium stearylfumerate, a hydrocarbon such as mineral oil, or hydrogenated vegetableoil such as hydrogenated soybean oil (Sterotex®), higher fatty acids andtheir alkali-metal and alkaline earth metal salts, such as aluminum,calcium, magnesium, zinc, stearic acid, sodium stearates, glycerol,talc, waxes, Stearowet®, boric acid, sodium benzoate, sodium acetate,sodium chloride, leucine, a polyethylene glycol (e.g., PEG-4000) or amethoxypolyethylene glycol such as Carbowax™, sodium oleate, sodiumbenzoate, glyceryl behenate, polyethylene glycol, magnesium or sodiumlauryl sulfate, colloidal silica such as Syloid™, Cab-O-Sil®, a starchsuch as corn starch, silicone oil, a surfactant, and the like.

Plasticizers include compounds used to soften the microencapsulationmaterial or film coatings to make them less brittle. Suitableplasticizers include, e.g., polyethylene glycols such as PEG 300, PEG400, PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid, propyleneglycol, oleic acid, triethyl cellulose and triacetin. Plasticizers canalso function as dispersing agents or wetting agents.

Solubilizers include compounds such as triacetin, triethylcitrate, ethyloleate, ethyl caprylate, sodium lauryl sulfate, sodium doccusate,vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone,N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethylcellulose, hydroxypropyl cyclodextrins, ethanol, n-butanol, isopropylalcohol, cholesterol, bile salts, polyethylene glycol 200-600,glycofurol, transcutol, propylene glycol, dimethyl isosorbide, and thelike.

Stabilizers include compounds such as any antioxidation agents, buffers,acids, preservatives and the like.

Suspending agents include compounds such as polyvinylpyrrolidone, e.g.,polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidoneK25, or polyvinylpyrrolidone K30, vinyl pyrrolidone/vinyl acetatecopolymer (S630), polyethylene glycol, e.g., the polyethylene glycol canhave a molecular weight of about 300 to about 6000, or about 3350 toabout 4000, or about 7000 to about 5400, sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose, hydroxymethylcelluloseacetate stearate, polysorbate-80, hydroxyethylcellulose, sodiumalginate, gums, such as, e.g., gum tragacanth and gum acacia, guar gum,xanthans, including xanthan gum, sugars, cellulosics, such as, e.g.,sodium carboxymethylcellulose, methylcellulose, sodiumcarboxymethylcellulose, hydroxypropylmethylcellulose,hydroxyethylcellulose, polysorbate-80, sodium alginate, polyethoxylatedsorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone andthe like.

Surfactants include compounds such as sodium lauryl sulfate, sodiumdocusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitanmonooleate, polyoxyethylene sorbitan monooleate, polysorbates,polaxomers, bile salts, glyceryl monostearate, copolymers of ethyleneoxide and propylene oxide, e.g., Pluronic® (BASF), and the like.Additional surfactants include polyoxyethylene fatty acid glycerides andvegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil; andpolyoxyethylene alkylethers and alkylphenyl ethers, e.g., octoxynol 10,octoxynol 40. Sometimes, surfactants is included to enhance physicalstability or for other purposes.

Viscosity enhancing agents include, e.g., methyl cellulose, xanthan gum,carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, hydroxypropylmethyl cellulose acetate stearate,hydroxypropylmethyl cellulose phthalate, carbomer, polyvinyl alcohol,alginates, acacia, chitosans and combinations thereof.

Wetting agents include compounds such as oleic acid, glycerylmonostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamineoleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitanmonolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate,sodium doccusate, triacetin, Tween 80, vitamin E TPGS, ammonium saltsand the like.

Therapeutic Regimens

In some embodiments, the pharmaceutical compositions described hereinare administered for therapeutic applications. In some embodiments, thepharmaceutical composition is administered once per day, twice per day,three times per day or more. The pharmaceutical composition isadministered daily, every day, every alternate day, five days a week,once a week, every other week, two weeks per month, three weeks permonth, once a month, twice a month, three times per month, or more. Thepharmaceutical composition is administered for at least 1 month, 2months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9months, 10 months, 11 months, 12 months, 18 months, 2 years, 3 years, ormore.

In some embodiments, one or more pharmaceutical compositions areadministered simutaneously, sequentially, or at an interval period oftime. In some embodiments, one or more pharmaceutical compositions areadministered simutaneously. In some cases, one or more pharmaceuticalcompositions are administered sequentially. In additional cases, one ormore pharmaceutical compositions are administered at an interval periodof time (e.g., the first administration of a first pharmaceuticalcomposition is on day one followed by an interval of at least 1, 2, 3,4, 5, or more days prior to the administration of at least a secondpharmaceutical composition).

In some embodiments, two or more different pharmaceutical compositionsare coadministered. In some instances, the two or more differentpharmaceutical compositions are coadministered simutaneously. In somecases, the two or more different pharmaceutical compositions arecoadministered sequentially without a gap of time betweenadministrations. In other cases, the two or more differentpharmaceutical compositions are coadministered sequentially with a gapof about 0.5 hour, 1 hour, 2 hour, 3 hour, 12 hours, 1 day, 2 days, ormore between administrations.

In the case wherein the patient's status does improve, upon the doctor'sdiscretion the administration of the composition is given continuously;alternatively, the dose of the composition being administered istemporarily reduced or temporarily suspended for a certain length oftime (i.e., a “drug holiday”). In some instances, the length of the drugholiday varies between 2 days and 1 year, including by way of exampleonly, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days,15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320days, 350 days, or 365 days. The dose reduction during a drug holiday isfrom 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100%.

Once improvement of the patient's conditions has occurred, a maintenancedose is administered if necessary. Subsequently, the dosage or thefrequency of administration, or both, are optionally reduced, as afunction of the symptoms, to a level at which the improved disease,disorder or condition is retained.

In some embodiments, the amount of a given agent that correspond to suchan amount varies depending upon factors such as the particular compound,the severity of the disease, the identity (e.g., weight) of the subjector host in need of treatment, but nevertheless is routinely determinedin a manner known in the art according to the particular circumstancessurrounding the case, including, e.g., the specific agent beingadministered, the route of administration, and the subject or host beingtreated. In some instances, the desired dose is conveniently presentedin a single dose or as divided doses administered simultaneously (orover a short period of time) or at appropriate intervals, for example astwo, three, four or more sub-doses per day.

The foregoing ranges are merely suggestive, as the number of variablesin regard to an individual treatment regime is large, and considerableexcursions from these recommended values are not uncommon. Such dosagesare altered depending on a number of variables, not limited to theactivity of the compound used, the disease or condition to be treated,the mode of administration, the requirements of the individual subject,the severity of the disease or condition being treated, and the judgmentof the practitioner.

In some embodiments, toxicity and therapeutic efficacy of suchtherapeutic regimens are determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, including, but notlimited to, the determination of the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between the toxic and therapeuticeffects is the therapeutic index and it is expressed as the ratiobetween LD50 and ED50. Compounds exhibiting high therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesare used in formulating a range of dosage for use in human. The dosageof such compounds lies preferably within a range of circulatingconcentrations that include the ED50 with minimal toxicity. The dosagevaries within this range depending upon the dosage form employed and theroute of administration utilized.

Kits/Article of Manufacture

Disclosed herein, in certain embodiments, are kits and articles ofmanufacture for use with one or more of the compositions and methodsdescribed herein. Such kits include a carrier, package, or containerthat is compartmentalized to receive one or more containers such asvials, tubes, and the like, each of the container(s) comprising one ofthe separate elements to be used in a method described herein. Suitablecontainers include, for example, bottles, vials, syringes, and testtubes. In one embodiment, the containers are formed from a variety ofmaterials such as glass or plastic.

The articles of manufacture provided herein contain packaging materials.Examples of pharmaceutical packaging materials include, but are notlimited to, blister packs, bottles, tubes, bags, containers, bottles,and any packaging material suitable for a selected formulation andintended mode of administration and treatment.

For example, the container(s) include a molecule of Formula (I):A-X—B—Y—C, optionally conjugated to an endosomolytic moiety D asdisclosed herein. Such kits optionally include an identifyingdescription or label or instructions relating to its use in the methodsdescribed herein.

A kit typically includes labels listing contents and/or instructions foruse and package inserts with instructions for use. A set of instructionswill also typically be included.

In one embodiment, a label is on or associated with the container. Inone embodiment, a label is on a container when letters, numbers, orother characters forming the label are attached, molded or etched intothe container itself; a label is associated with a container when it ispresent within a receptacle or carrier that also holds the container,e.g., as a package insert. In one embodiment, a label is used toindicate that the contents are to be used for a specific therapeuticapplication. The label also indicates directions for use of thecontents, such as in the methods described herein.

In certain embodiments, the pharmaceutical compositions are presented ina pack or dispenser device which contains one or more unit dosage formscontaining a compound provided herein. The pack, for example, containsmetal or plastic foil, such as a blister pack. In one embodiment, thepack or dispenser device is accompanied by instructions foradministration. In one embodiment, the pack or dispenser is alsoaccompanied with a notice associated with the container in formprescribed by a governmental agency regulating the manufacture, use, orsale of pharmaceuticals, which notice is reflective of approval by theagency of the form of the drug for human or veterinary administration.Such notice, for example, is the labeling approved by the U.S. Food andDrug Administration for prescription drugs, or the approved productinsert. In one embodiment, compositions containing a compound providedherein formulated in a compatible pharmaceutical carrier are alsoprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition.

Certain Terminology

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the claimed subject matter belongs. It is to be understoodthat the foregoing general description and the following detaileddescription are exemplary and explanatory only and are not restrictiveof any subject matter claimed. In this application, the use of thesingular includes the plural unless specifically stated otherwise. Itmust be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. In this application, theuse of “or” means “and/or” unless stated otherwise. Furthermore, use ofthe term “including” as well as other forms, such as “include”,“includes,” and “included,” is not limiting.

As used herein, ranges and amounts can be expressed as “about” aparticular value or range. About also includes the exact amount. Hence“about 5 μL” means “about 5 μL” and also “5 μL.” Generally, the term“about” includes an amount that is expected to be within experimentalerror.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

As used herein, the terms “individual(s)”, “subject(s)” and “patient(s)”mean any mammal. In some embodiments, the mammal is a human. In someembodiments, the mammal is a non-human. None of the terms require or arelimited to situations characterized by the supervision (e.g. constant orintermittent) of a health care worker (e.g. a doctor, a registerednurse, a nurse practitioner, a physician's assistant, an orderly or ahospice worker).

EXAMPLES

These examples are provided for illustrative purposes only and not tolimit the scope of the claims provided herein.

Example 1. Sequences

Tables 1, 4, 7, 8, and 10 illustrate target sequences described herein.Tables 2, 3, 5, 6, 9, 11, and 12 illustrate polynucleic acid moleculesequences described herein.

TABLE 1 KRAS Target Sequences sequence position in SEQ Id # NM_033360.2target site in NM_033360.2 ID NO: 182 182-200 AAAUGACUGAAUAUAAACUUGUG 1183 183-201 AAUGACUGAAUAUAAACUUGUGG 2 197 197-215AACUUGUGGUAGUUGGAGCUGGU 3 224 224-242 UAGGCAAGAGUGCCUUGACGAUA 4 226226-244 GGCAAGAGUGCCUUGACGAUACA 5 227 227-245 GCAAGAGUGCCUUGACGAUACAG 6228 228-246 CAAGAGUGCCUUGACGAUACAGC 7 232 232-250AGUGCCUUGACGAUACAGCUAAU 8 233 233-251 GUGCCUUGACGAUACAGCUAAUU 9 236236-254 CCUUGACGAUACAGCUAAUUCAG 10 237 237-255 CUUGACGAUACAGCUAAUUCAGA11 245 245-263 UACAGCUAAUUCAGAAUCAUUUU 12 266 266-284UUGUGGACGAAUAUGAUCCAACA 13 269 269-287 UGGACGAAUAUGAUCCAACAAUA 14 270270-288 GGACGAAUAUGAUCCAACAAUAG 15

TABLE 2 KRAS siRNA sequences sequence SEQ SEQ position in ID antisensestrand sequence ID Id # NM_033360.2 sense strand sequence (5′-3′) NO:(5′-3′) NO: 182 182-200 AUGACUGAAUAUAAACU 16 CAAGUUUAUAUUCAG 17 UGTTUCAUTT 183 183-201 UGACUGAAUAUAAACUU 18 ACAAGUUUAUAUUCA 19 GUTT GUCATT197 197-215 CUUGUGGUAGUUGGAGC 20 CAGCUCCAACUACCAC 21 UGTT AAGTT 224224-242 GGCAAGAGUGCCUUGAC 22 UCGUCAAGGCACUCU 23 GATT UGCCTT 226 226-244CAAGAGUGCCUUGACGA 24 UAUCGUCAAGGCACU 25 UATT CUUGTT 227 227-245AAGAGUGCCUUGACGAU 26 GUAUCGUCAAGGCAC 27 ACTT UCUUTT 228 228-246AGAGUGCCUUGACGAUA 28 UGUAUCGUCAAGGCA 29 CATT CUCUTT 232 232-250UGCCUUGACGAUACAGC 30 UAGCUGUAUCGUCAA 31 UATT GGCATT 233 233-251GCCUUGACGAUACAGCU 32 UUAGCUGUAUCGUCA 33 AATT AGGCTT 236 236-254UUGACGAUACAGCUAAU 34 GAAUUAGCUGUAUCG 35 UCTT UCAATT 237 237-255UGACGAUACAGCUAAUU 36 UGAAUUAGCUGUAUC 37 CATT GUCATT 245 245-263CAGCUAAUUCAGAAUCA 38 AAUGAUUCUGAAUUA 39 UUTT GCUGTT 266 266-284GUGGACGAAUAUGAUCC 40 UUGGAUCAUAUUCGU 41 AATT CCACTT 269 269-287GACGAAUAUGAUCCAAC 42 UUGUUGGAUCAUAUU 43 AATT CGUCTT 270 270-288ACGAAUAUGAUCCAACA 44 AUUGUUGGAUCAUAU 45 AUTT UCGUTT

TABLE 3 KRAS siRNA Sequences with Chemical Modification siRNA sequencewith siRNA sequence with chemical modification SEQ chemical modificationSEQ sequence position sense strand sequence ID antisense strand sequence(5′- ID Id # in NM_033360.2 (5′-3′) NO: 3′) NO: 182 182-200auGfaCfuGfaAfuAfuAf 46 CfAfaGfuUfuAfuAfuUfcAfgU 47 aAfcUfuGfdTsdTfcAfudTsdT 183 183-201 ugAfcUfgAfaUfaUfaAf 48 AfCfaAfgUfuUfaUfaUfuCfaGf49 aCfuUfgUfdTsdT uCfadTsdT 197 197-215 cuUfgUfgGfuAfgUfuGf 50CfAfgCfuCfcAfaCfuAfcCfaCf 51 gAfgCfuGfdTsdT aAfgdTsdT 224 224-242ggCfaAfgAfgUfgCfcUf 52 UfCfgUfcAfaGfgCfaCfuCfuUf 53 uGfaCfgAfdTsdTgCfcdTsdT 226 226-244 caAfgAfgUfgCfcUfuGf 54 UfAfuCfgUfcAfaGfgCfaCfuCf55 aCfgAfuAfdTsdT uUfgdTsdT 227 227-245 aaGfaGfuGfcCfuUfgAf 56GfUfaUfcGfuCfaAfgGfcAfcUf 57 cGfaUfaCfdTsdT cUfudTsdT 228 228-246agAfgUfgCfcUfuGfaCf 58 UfGfuAfuCfgUfcAfaGfgCfaCf 59 gAfuAfcAfdTsdTuCfudTsdT 232 232-250 ugCfcUfuGfaCfgAfuAf 60 UfAfgCfuGfuAfuCfgUfcAfaGf61 cAfgCfuAfdTsdT gCfadTsdT 233 233-251 gcCfuUfgAfcGfaUfaCf 62UfUfaGfcUfgUfaUfcGfuCfaAf 63 aGfcUfaAfdTsdT gGfcdTsdT 236 236-254uuGfaCfgAfuAfcAfgCf 64 GfAfaUfuAfgCfuGfuAfuCfgU 65 uAfaUfuCfdTsdTfcAfadTsdT 237 237-255 ugAfcGfaUfaCfaGfcUf 66 UfGfaAfuUfaGfcUfgUfaUfcGf67 aAfuUfcAfdTsdT uCfadTsdT 245 245-263 caGfcUfaAfuUfcAfgAf 68AfAfuGfaUfuCfuGfaAfuUfaGf 69 aUfcAfuUfdTsdT cUfgdTsdT 266 266-284guGfgAfcGfaAfuAfuGf 70 UfUfgGfaUfcAfuAfuUfcGfuCf 71 aUfcCfaAfdTsdTcAfcdTsdT 269 269-287 gaCfgAfaUfaUfgAfuCf 72 UfUfgUfuGfgAfuCfaUfaUfuCf73 cAfaCfaAfdTsdT gUfcdTsdT 270 270-288 acGfaAfuAfuGfaUfcCf 74AfUfuGfuUfgGfaUfcAfuAfuU 75 aAfcAfaUfdTsdT fcGfudTsdT siRNA Sequencewith Chemical Modification Info lower case (n) = 2′-O-Me; Nf = 2′-F; dT= deoxy-T residue; s = phosphorothioate backbone modification; iB= inverted abasic

TABLE 4 EGFR Target Sequences hs 19mer pos. in sequence of total 23merSEQ Id # NM_005228.3 target site in NM_005228.3 ID NO: 68 68-86GGCGGCCGGAGUCCCGAGCUAGC 76 71 71-89 GGCCGGAGUCCCGAGCUAGCCCC 77 72 72-90GCCGGAGUCCCGAGCUAGCCCCG 78 73 73-91 CCGGAGUCCCGAGCUAGCCCCGG 79 74 74-92CGGAGUCCCGAGCUAGCCCCGGC 80 75 75-93 GGAGUCCCGAGCUAGCCCCGGCG 81 76 76-94GAGUCCCGAGCUAGCCCCGGCGG 82 78 78-96 GUCCCGAGCUAGCCCCGGCGGCC 83 114114-132 CCGGACGACAGGCCACCUCGUCG 84 115 115-133 CGGACGACAGGCCACCUCGUCGG85 116 116-134 GGACGACAGGCCACCUCGUCGGC 86 117 117-135GACGACAGGCCACCUCGUCGGCG 87 118 118-136 ACGACAGGCCACCUCGUCGGCGU 88 120120-138 GACAGGCCACCUCGUCGGCGUCC 89 121 121-139 ACAGGCCACCUCGUCGGCGUCCG90 122 122-140 CAGGCCACCUCGUCGGCGUCCGC 91 123 123-141AGGCCACCUCGUCGGCGUCCGCC 92 124 124-142 GGCCACCUCGUCGGCGUCCGCCC 93 125125-143 GCCACCUCGUCGGCGUCCGCCCG 94 126 126-144 CCACCUCGUCGGCGUCCGCCCGA95 127 127-145 CACCUCGUCGGCGUCCGCCCGAG 96 128 128-146ACCUCGUCGGCGUCCGCCCGAGU 97 129 129-147 CCUCGUCGGCGUCCGCCCGAGUC 98 130130-148 CUCGUCGGCGUCCGCCCGAGUCC 99 131 131-149 UCGUCGGCGUCCGCCCGAGUCCC100 132 132-150 CGUCGGCGUCCGCCCGAGUCCCC 101 135 135-153CGGCGUCCGCCCGAGUCCCCGCC 102 136 136-154 GGCGUCCGCCCGAGUCCCCGCCU 103 141141-159 CCGCCCGAGUCCCCGCCUCGCCG 104 164 164-182 CCAACGCCACAACCACCGCGCAC105 165 165-183 CAACGCCACAACCACCGCGCACG 106 166 166-184AACGCCACAACCACCGCGCACGG 107 168 168-186 CGCCACAACCACCGCGCACGGCC 108 169169-187 GCCACAACCACCGCGCACGGCCC 109 170 170-188 CCACAACCACCGCGCACGGCCCC110 247 247-265 CGAUGCGACCCUCCGGGACGGCC 111 248 248-266GAUGCGACCCUCCGGGACGGCCG 112 249 249-267 AUGCGACCCUCCGGGACGGCCGG 113 251251-269 GCGACCCUCCGGGACGGCCGGGG 114 252 252-270 CGACCCUCCGGGACGGCCGGGGC115 254 254-272 ACCCUCCGGGACGGCCGGGGCAG 116 329 329-347AAAGAAAGUUUGCCAAGGCACGA 117 330 330-348 AAGAAAGUUUGCCAAGGCACGAG 118 332332-350 GAAAGUUUGCCAAGGCACGAGUA 119 333 333-351 AAAGUUUGCCAAGGCACGAGUAA120 334 334-352 AAGUUUGCCAAGGCACGAGUAAC 121 335 335-353AGUUUGCCAAGGCACGAGUAACA 122 336 336-354 GUUUGCCAAGGCACGAGUAACAA 123 337337-355 UUUGCCAAGGCACGAGUAACAAG 124 338 338-356 UUGCCAAGGCACGAGUAACAAGC125 361 361-379 UCACGCAGUUGGGCACUUUUGAA 126 362 362-380CACGCAGUUGGGCACUUUUGAAG 127 363 363-381 ACGCAGUUGGGCACUUUUGAAGA 128 364364-382 CGCAGUUGGGCACUUUUGAAGAU 129 365 365-383 GCAGUUGGGCACUUUUGAAGAUC130 366 366-384 CAGUUGGGCACUUUUGAAGAUCA 131 367 367-385AGUUGGGCACUUUUGAAGAUCAU 132 368 368-386 GUUGGGCACUUUUGAAGAUCAUU 133 369369-387 UUGGGCACUUUUGAAGAUCAUUU 134 377 377-395 UUUUGAAGAUCAUUUUCUCAGCC135 379 379-397 UUGAAGAUCAUUUUCUCAGCCUC 136 380 380-398UGAAGAUCAUUUUCUCAGCCUCC 137 385 385-403 AUCAUUUUCUCAGCCUCCAGAGG 138 394394-412 UCAGCCUCCAGAGGAUGUUCAAU 139 396 396-414 AGCCUCCAGAGGAUGUUCAAUAA140 397 397-415 GCCUCCAGAGGAUGUUCAAUAAC 141 401 401-419CCAGAGGAUGUUCAAUAACUGUG 142 403 403-421 AGAGGAUGUUCAAUAACUGUGAG 143 407407-425 GAUGUUCAAUAACUGUGAGGUGG 144 409 409-427 UGUUCAAUAACUGUGAGGUGGUC145 410 410-428 GUUCAAUAACUGUGAGGUGGUCC 146 411 411-429UUCAAUAACUGUGAGGUGGUCCU 147 412 412-430 UCAAUAACUGUGAGGUGGUCCUU 148 413413-431 CAAUAACUGUGAGGUGGUCCUUG 149 414 414-432 AAUAACUGUGAGGUGGUCCUUGG150 416 416-434 UAACUGUGAGGUGGUCCUUGGGA 151 418 418-436ACUGUGAGGUGGUCCUUGGGAAU 152 419 419-437 CUGUGAGGUGGUCCUUGGGAAUU 153 425425-443 GGUGGUCCUUGGGAAUUUGGAAA 154 431 431-449 CCUUGGGAAUUUGGAAAUUACCU155 432 432-450 CUUGGGAAUUUGGAAAUUACCUA 156 433 433-451UUGGGAAUUUGGAAAUUACCUAU 157 434 434-452 UGGGAAUUUGGAAAUUACCUAUG 158 458458-476 GCAGAGGAAUUAUGAUCUUUCCU 159 459 459-477 CAGAGGAAUUAUGAUCUUUCCUU160 463 463-481 GGAAUUAUGAUCUUUCCUUCUUA 161 464 464-482GAAUUAUGAUCUUUCCUUCUUAA 162 466 466-484 AUUAUGAUCUUUCCUUCUUAAAG 163 468468-486 UAUGAUCUUUCCUUCUUAAAGAC 164 471 471-489 GAUCUUUCCUUCUUAAAGACCAU165 476 476-494 UUCCUUCUUAAAGACCAUCCAGG 166 477 477-495UCCUUCUUAAAGACCAUCCAGGA 167 479 479-497 CUUCUUAAAGACCAUCCAGGAGG 168 481481-499 UCUUAAAGACCAUCCAGGAGGUG 169 482 482-500 CUUAAAGACCAUCCAGGAGGUGG170 492 492-510 AUCCAGGAGGUGGCUGGUUAUGU 171 493 493-511UCCAGGAGGUGGCUGGUUAUGUC 172 494 494-512 CCAGGAGGUGGCUGGUUAUGUCC 173 495495-513 CAGGAGGUGGCUGGUUAUGUCCU 174 496 496-514 AGGAGGUGGCUGGUUAUGUCCUC175 497 497-515 GGAGGUGGCUGGUUAUGUCCUCA 176 499 499-517AGGUGGCUGGUUAUGUCCUCAUU 177 520 520-538 UUGCCCUCAACACAGUGGAGCGA 178 542542-560 AAUUCCUUUGGAAAACCUGCAGA 179 543 543-561 AUUCCUUUGGAAAACCUGCAGAU180 550 550-568 UGGAAAACCUGCAGAUCAUCAGA 181 551 551-569GGAAAACCUGCAGAUCAUCAGAG 182 553 553-571 AAAACCUGCAGAUCAUCAGAGGA 183 556556-574 ACCUGCAGAUCAUCAGAGGAAAU 184 586 586-604 ACGAAAAUUCCUAUGCCUUAGCA185 587 587-605 CGAAAAUUCCUAUGCCUUAGCAG 186 589 589-607AAAAUUCCUAUGCCUUAGCAGUC 187 592 592-610 AUUCCUAUGCCUUAGCAGUCUUA 188 593593-611 UUCCUAUGCCUUAGCAGUCUUAU 189 594 594-612 UCCUAUGCCUUAGCAGUCUUAUC190 596 596-614 CUAUGCCUUAGCAGUCUUAUCUA 191 597 597-615UAUGCCUUAGCAGUCUUAUCUAA 192 598 598-616 AUGCCUUAGCAGUCUUAUCUAAC 193 599599-617 UGCCUUAGCAGUCUUAUCUAACU 194 600 600-618 GCCUUAGCAGUCUUAUCUAACUA195 601 601-619 CCUUAGCAGUCUUAUCUAACUAU 196 602 602-620CUUAGCAGUCUUAUCUAACUAUG 197 603 603-621 UUAGCAGUCUUAUCUAACUAUGA 198 604604-622 UAGCAGUCUUAUCUAACUAUGAU 199 605 605-623 AGCAGUCUUAUCUAACUAUGAUG200 608 608-626 AGUCUUAUCUAACUAUGAUGCAA 201 609 609-627GUCUUAUCUAACUAUGAUGCAAA 202 610 610-628 UCUUAUCUAACUAUGAUGCAAAU 203 611611-629 CUUAUCUAACUAUGAUGCAAAUA 204 612 612-630 UUAUCUAACUAUGAUGCAAAUAA205 613 613-631 UAUCUAACUAUGAUGCAAAUAAA 206 614 614-632AUCUAACUAUGAUGCAAAUAAAA 207 616 616-634 CUAACUAUGAUGCAAAUAAAACC 208 622622-640 AUGAUGCAAAUAAAACCGGACUG 209 623 623-641 UGAUGCAAAUAAAACCGGACUGA210 624 624-642 GAUGCAAAUAAAACCGGACUGAA 211 626 626-644UGCAAAUAAAACCGGACUGAAGG 212 627 627-645 GCAAAUAAAACCGGACUGAAGGA 213 628628-646 CAAAUAAAACCGGACUGAAGGAG 214 630 630-648 AAUAAAACCGGACUGAAGGAGCU215 631 631-649 AUAAAACCGGACUGAAGGAGCUG 216 632 632-650UAAAACCGGACUGAAGGAGCUGC 217 633 633-651 AAAACCGGACUGAAGGAGCUGCC 218 644644-662 GAAGGAGCUGCCCAUGAGAAAUU 219 665 665-683 UUUACAGGAAAUCCUGCAUGGCG220 668 668-686 ACAGGAAAUCCUGCAUGGCGCCG 221 669 669-687CAGGAAAUCCUGCAUGGCGCCGU 222 670 670-688 AGGAAAUCCUGCAUGGCGCCGUG 223 671671-689 GGAAAUCCUGCAUGGCGCCGUGC 224 672 672-690 GAAAUCCUGCAUGGCGCCGUGCG225 674 674-692 AAUCCUGCAUGGCGCCGUGCGGU 226 676 676-694UCCUGCAUGGCGCCGUGCGGUUC 227 677 677-695 CCUGCAUGGCGCCGUGCGGUUCA 228 678678-696 CUGCAUGGCGCCGUGCGGUUCAG 229 680 680-698 GCAUGGCGCCGUGCGGUUCAGCA230 681 681-699 CAUGGCGCCGUGCGGUUCAGCAA 231 682 682-700AUGGCGCCGUGCGGUUCAGCAAC 232 683 683-701 UGGCGCCGUGCGGUUCAGCAACA 233 684684-702 GGCGCCGUGCGGUUCAGCAACAA 234 685 685-703 GCGCCGUGCGGUUCAGCAACAAC235 686 686-704 CGCCGUGCGGUUCAGCAACAACC 236 688 688-706CCGUGCGGUUCAGCAACAACCCU 237 690 690-708 GUGCGGUUCAGCAACAACCCUGC 238 692692-710 GCGGUUCAGCAACAACCCUGCCC 239 698 698-716 CAGCAACAACCCUGCCCUGUGCA240 700 700-718 GCAACAACCCUGCCCUGUGCAAC 241 719 719-737CAACGUGGAGAGCAUCCAGUGGC 242 720 720-738 AACGUGGAGAGCAUCCAGUGGCG 243 721721-739 ACGUGGAGAGCAUCCAGUGGCGG 244 724 724-742 UGGAGAGCAUCCAGUGGCGGGAC245 725 725-743 GGAGAGCAUCCAGUGGCGGGACA 246 726 726-744GAGAGCAUCCAGUGGCGGGACAU 247 733 733-751 UCCAGUGGCGGGACAUAGUCAGC 248 734734-752 CCAGUGGCGGGACAUAGUCAGCA 249 736 736-754 AGUGGCGGGACAUAGUCAGCAGU250 737 737-755 GUGGCGGGACAUAGUCAGCAGUG 251 763 763-781UUCUCAGCAACAUGUCGAUGGAC 252 765 765-783 CUCAGCAACAUGUCGAUGGACUU 253 766766-784 UCAGCAACAUGUCGAUGGACUUC 254 767 767-785 CAGCAACAUGUCGAUGGACUUCC255 769 769-787 GCAACAUGUCGAUGGACUUCCAG 256 770 770-788CAACAUGUCGAUGGACUUCCAGA 257 771 771-789 AACAUGUCGAUGGACUUCCAGAA 258 772772-790 ACAUGUCGAUGGACUUCCAGAAC 259 775 775-793 UGUCGAUGGACUUCCAGAACCAC260 789 789-807 CAGAACCACCUGGGCAGCUGCCA 261 798 798-816CUGGGCAGCUGCCAAAAGUGUGA 262 800 800-818 GGGCAGCUGCCAAAAGUGUGAUC 263 805805-823 GCUGCCAAAAGUGUGAUCCAAGC 264 806 806-824 CUGCCAAAAGUGUGAUCCAAGCU265 807 807-825 UGCCAAAAGUGUGAUCCAAGCUG 266 810 810-828CAAAAGUGUGAUCCAAGCUGUCC 267 814 814-832 AGUGUGAUCCAAGCUGUCCCAAU 268 815815-833 GUGUGAUCCAAGCUGUCCCAAUG 269 817 817-835 GUGAUCCAAGCUGUCCCAAUGGG270 818 818-836 UGAUCCAAGCUGUCCCAAUGGGA 271 819 819-837GAUCCAAGCUGUCCCAAUGGGAG 272 820 820-838 AUCCAAGCUGUCCCAAUGGGAGC 273 821821-839 UCCAAGCUGUCCCAAUGGGAGCU 274 823 823-841 CAAGCUGUCCCAAUGGGAGCUGC275 826 826-844 GCUGUCCCAAUGGGAGCUGCUGG 276 847 847-865GGGGUGCAGGAGAGGAGAACUGC 277 871 871-889 AGAAACUGACCAAAAUCAUCUGU 278 872872-890 GAAACUGACCAAAAUCAUCUGUG 279 873 873-891 AAACUGACCAAAAUCAUCUGUGC280 877 877-895 UGACCAAAAUCAUCUGUGCCCAG 281 878 878-896GACCAAAAUCAUCUGUGCCCAGC 282 881 881-899 CAAAAUCAUCUGUGCCCAGCAGU 283 890890-908 CUGUGCCCAGCAGUGCUCCGGGC 284 892 892-910 GUGCCCAGCAGUGCUCCGGGCGC285 929 929-947 CCCCAGUGACUGCUGCCACAACC 286 930 930-948CCCAGUGACUGCUGCCACAACCA 287 979 979-997 GGGAGAGCGACUGCCUGGUCUGC 288 980980-998 GGAGAGCGACUGCCUGGUCUGCC 289 981 981-999 GAGAGCGACUGCCUGGUCUGCCG290 982  982-1000 AGAGCGACUGCCUGGUCUGCCGC 291 983  983-1001GAGCGACUGCCUGGUCUGCCGCA 292 984  984-1002 AGCGACUGCCUGGUCUGCCGCAA 293989  989-1007 CUGCCUGGUCUGCCGCAAAUUCC 294 990  990-1008UGCCUGGUCUGCCGCAAAUUCCG 295 991  991-1009 GCCUGGUCUGCCGCAAAUUCCGA 296992  992-1010 CCUGGUCUGCCGCAAAUUCCGAG 297 994  994-1012UGGUCUGCCGCAAAUUCCGAGAC 298 995  995-1013 GGUCUGCCGCAAAUUCCGAGACG 299996  996-1014 GUCUGCCGCAAAUUCCGAGACGA 300 997  997-1015UCUGCCGCAAAUUCCGAGACGAA 301 999  999-1017 UGCCGCAAAUUCCGAGACGAAGC 3021004 1004-1022 CAAAUUCCGAGACGAAGCCACGU 303 1005 1005-1023AAAUUCCGAGACGAAGCCACGUG 304 1006 1006-1024 AAUUCCGAGACGAAGCCACGUGC 3051007 1007-1025 AUUCCGAGACGAAGCCACGUGCA 306 1008 1008-1026UUCCGAGACGAAGCCACGUGCAA 307 1010 1010-1028 CCGAGACGAAGCCACGUGCAAGG 3081013 1013-1031 AGACGAAGCCACGUGCAAGGACA 309 1014 1014-1032GACGAAGCCACGUGCAAGGACAC 310 1015 1015-1033 ACGAAGCCACGUGCAAGGACACC 3111016 1016-1034 CGAAGCCACGUGCAAGGACACCU 312 1040 1040-1058CCCCCCACUCAUGCUCUACAACC 313 1042 1042-1060 CCCCACUCAUGCUCUACAACCCC 3141044 1044-1062 CCACUCAUGCUCUACAACCCCAC 315 1047 1047-1065CUCAUGCUCUACAACCCCACCAC 316 1071 1071-1089 UACCAGAUGGAUGUGAACCCCGA 3171073 1073-1091 CCAGAUGGAUGUGAACCCCGAGG 318 1074 1074-1092CAGAUGGAUGUGAACCCCGAGGG 319 1075 1075-1093 AGAUGGAUGUGAACCCCGAGGGC 3201077 1077-1095 AUGGAUGUGAACCCCGAGGGCAA 321 1078 1078-1096UGGAUGUGAACCCCGAGGGCAAA 322 1080 1080-1098 GAUGUGAACCCCGAGGGCAAAUA 3231084 1084-1102 UGAACCCCGAGGGCAAAUACAGC 324 1085 1085-1103GAACCCCGAGGGCAAAUACAGCU 325 1087 1087-1105 ACCCCGAGGGCAAAUACAGCUUU 3261088 1088-1106 CCCCGAGGGCAAAUACAGCUUUG 327 1089 1089-1107CCCGAGGGCAAAUACAGCUUUGG 328 1096 1096-1114 GCAAAUACAGCUUUGGUGCCACC 3291097 1097-1115 CAAAUACAGCUUUGGUGCCACCU 330 1098 1098-1116AAAUACAGCUUUGGUGCCACCUG 331 1104 1104-1122 AGCUUUGGUGCCACCUGCGUGAA 3321106 1106-1124 CUUUGGUGCCACCUGCGUGAAGA 333 1112 1112-1130UGCCACCUGCGUGAAGAAGUGUC 334 1116 1116-1134 ACCUGCGUGAAGAAGUGUCCCCG 3351117 1117-1135 CCUGCGUGAAGAAGUGUCCCCGU 336 1118 1118-1136CUGCGUGAAGAAGUGUCCCCGUA 337 1119 1119-1137 UGCGUGAAGAAGUGUCCCCGUAA 3381120 1120-1138 GCGUGAAGAAGUGUCCCCGUAAU 339 1121 1121-1139CGUGAAGAAGUGUCCCCGUAAUU 340 1122 1122-1140 GUGAAGAAGUGUCCCCGUAAUUA 3411123 1123-1141 UGAAGAAGUGUCCCCGUAAUUAU 342 1124 1124-1142GAAGAAGUGUCCCCGUAAUUAUG 343 1125 1125-1143 AAGAAGUGUCCCCGUAAUUAUGU 3441126 1126-1144 AGAAGUGUCCCCGUAAUUAUGUG 345 1127 1127-1145GAAGUGUCCCCGUAAUUAUGUGG 346 1128 1128-1146 AAGUGUCCCCGUAAUUAUGUGGU 3471129 1129-1147 AGUGUCCCCGUAAUUAUGUGGUG 348 1130 1130-1148GUGUCCCCGUAAUUAUGUGGUGA 349 1132 1132-1150 GUCCCCGUAAUUAUGUGGUGACA 3501134 1134-1152 CCCCGUAAUUAUGUGGUGACAGA 351 1136 1136-1154CCGUAAUUAUGUGGUGACAGAUC 352 1137 1137-1155 CGUAAUUAUGUGGUGACAGAUCA 3531138 1138-1156 GUAAUUAUGUGGUGACAGAUCAC 354 1139 1139-1157UAAUUAUGUGGUGACAGAUCACG 355 1140 1140-1158 AAUUAUGUGGUGACAGAUCACGG 3561142 1142-1160 UUAUGUGGUGACAGAUCACGGCU 357 1145 1145-1163UGUGGUGACAGAUCACGGCUCGU 358 1147 1147-1165 UGGUGACAGAUCACGGCUCGUGC 3591148 1148-1166 GGUGACAGAUCACGGCUCGUGCG 360 1149 1149-1167GUGACAGAUCACGGCUCGUGCGU 361 1150 1150-1168 UGACAGAUCACGGCUCGUGCGUC 3621151 1151-1169 GACAGAUCACGGCUCGUGCGUCC 363 1152 1152-1170ACAGAUCACGGCUCGUGCGUCCG 364 1153 1153-1171 CAGAUCACGGCUCGUGCGUCCGA 3651154 1154-1172 AGAUCACGGCUCGUGCGUCCGAG 366 1155 1155-1173GAUCACGGCUCGUGCGUCCGAGC 367 1156 1156-1174 AUCACGGCUCGUGCGUCCGAGCC 3681157 1157-1175 UCACGGCUCGUGCGUCCGAGCCU 369 1160 1160-1178CGGCUCGUGCGUCCGAGCCUGUG 370 1200 1200-1218 AUGGAGGAAGACGGCGUCCGCAA 3711201 1201-1219 UGGAGGAAGACGGCGUCCGCAAG 372 1203 1203-1221GAGGAAGACGGCGUCCGCAAGUG 373 1204 1204-1222 AGGAAGACGGCGUCCGCAAGUGU 3741205 1205-1223 GGAAGACGGCGUCCGCAAGUGUA 375 1207 1207-1225AAGACGGCGUCCGCAAGUGUAAG 376 1208 1208-1226 AGACGGCGUCCGCAAGUGUAAGA 3771211 1211-1229 CGGCGUCCGCAAGUGUAAGAAGU 378 1212 1212-1230GGCGUCCGCAAGUGUAAGAAGUG 379 1213 1213-1231 GCGUCCGCAAGUGUAAGAAGUGC 3801214 1214-1232 CGUCCGCAAGUGUAAGAAGUGCG 381 1215 1215-1233GUCCGCAAGUGUAAGAAGUGCGA 382 1216 1216-1234 UCCGCAAGUGUAAGAAGUGCGAA 3831217 1217-1235 CCGCAAGUGUAAGAAGUGCGAAG 384 1219 1219-1237GCAAGUGUAAGAAGUGCGAAGGG 385 1220 1220-1238 CAAGUGUAAGAAGUGCGAAGGGC 3861221 1221-1239 AAGUGUAAGAAGUGCGAAGGGCC 387 1222 1222-1240AGUGUAAGAAGUGCGAAGGGCCU 388 1223 1223-1241 GUGUAAGAAGUGCGAAGGGCCUU 3891224 1224-1242 UGUAAGAAGUGCGAAGGGCCUUG 390 1225 1225-1243GUAAGAAGUGCGAAGGGCCUUGC 391 1226 1226-1244 UAAGAAGUGCGAAGGGCCUUGCC 3921229 1229-1247 GAAGUGCGAAGGGCCUUGCCGCA 393 1230 1230-1248AAGUGCGAAGGGCCUUGCCGCAA 394 1231 1231-1249 AGUGCGAAGGGCCUUGCCGCAAA 3951232 1232-1250 GUGCGAAGGGCCUUGCCGCAAAG 396 1233 1233-1251UGCGAAGGGCCUUGCCGCAAAGU 397 1235 1235-1253 CGAAGGGCCUUGCCGCAAAGUGU 3981236 1236-1254 GAAGGGCCUUGCCGCAAAGUGUG 399 1237 1237-1255AAGGGCCUUGCCGCAAAGUGUGU 400 1238 1238-1256 AGGGCCUUGCCGCAAAGUGUGUA 4011239 1239-1257 GGGCCUUGCCGCAAAGUGUGUAA 402 1241 1241-1259GCCUUGCCGCAAAGUGUGUAACG 403 1261 1261-1279 ACGGAAUAGGUAUUGGUGAAUUU 4041262 1262-1280 CGGAAUAGGUAUUGGUGAAUUUA 405 1263 1263-1281GGAAUAGGUAUUGGUGAAUUUAA 406 1264 1264-1282 GAAUAGGUAUUGGUGAAUUUAAA 4071266 1266-1284 AUAGGUAUUGGUGAAUUUAAAGA 408 1267 1267-1285UAGGUAUUGGUGAAUUUAAAGAC 409 1289 1289-1307 CUCACUCUCCAUAAAUGCUACGA 4101313 1313-1331 UAUUAAACACUUCAAAAACUGCA 411 1320 1320-1338CACUUCAAAAACUGCACCUCCAU 412 1321 1321-1339 ACUUCAAAAACUGCACCUCCAUC 4131322 1322-1340 CUUCAAAAACUGCACCUCCAUCA 414 1323 1323-1341UUCAAAAACUGCACCUCCAUCAG 415 1324 1324-1342 UCAAAAACUGCACCUCCAUCAGU 4161328 1328-1346 AAACUGCACCUCCAUCAGUGGCG 417 1332 1332-1350UGCACCUCCAUCAGUGGCGAUCU 418 1333 1333-1351 GCACCUCCAUCAGUGGCGAUCUC 4191335 1335-1353 ACCUCCAUCAGUGGCGAUCUCCA 420 1338 1338-1356UCCAUCAGUGGCGAUCUCCACAU 421 1344 1344-1362 AGUGGCGAUCUCCACAUCCUGCC 4221345 1345-1363 GUGGCGAUCUCCACAUCCUGCCG 423 1346 1346-1364UGGCGAUCUCCACAUCCUGCCGG 424 1347 1347-1365 GGCGAUCUCCACAUCCUGCCGGU 4251348 1348-1366 GCGAUCUCCACAUCCUGCCGGUG 426 1353 1353-1371CUCCACAUCCUGCCGGUGGCAUU 427 1354 1354-1372 UCCACAUCCUGCCGGUGGCAUUU 4281355 1355-1373 CCACAUCCUGCCGGUGGCAUUUA 429 1357 1357-1375ACAUCCUGCCGGUGGCAUUUAGG 430 1360 1360-1378 UCCUGCCGGUGGCAUUUAGGGGU 4311361 1361-1379 CCUGCCGGUGGCAUUUAGGGGUG 432 1362 1362-1380CUGCCGGUGGCAUUUAGGGGUGA 433 1363 1363-1381 UGCCGGUGGCAUUUAGGGGUGAC 4341366 1366-1384 CGGUGGCAUUUAGGGGUGACUCC 435 1369 1369-1387UGGCAUUUAGGGGUGACUCCUUC 436 1370 1370-1388 GGCAUUUAGGGGUGACUCCUUCA 4371371 1371-1389 GCAUUUAGGGGUGACUCCUUCAC 438 1372 1372-1390CAUUUAGGGGUGACUCCUUCACA 439 1373 1373-1391 AUUUAGGGGUGACUCCUUCACAC 4401374 1374-1392 UUUAGGGGUGACUCCUUCACACA 441 1404 1404-1422CCUCUGGAUCCACAGGAACUGGA 442 1408 1408-1426 UGGAUCCACAGGAACUGGAUAUU 4431409 1409-1427 GGAUCCACAGGAACUGGAUAUUC 444 1411 1411-1429AUCCACAGGAACUGGAUAUUCUG 445 1412 1412-1430 UCCACAGGAACUGGAUAUUCUGA 4461419 1419-1437 GAACUGGAUAUUCUGAAAACCGU 447 1426 1426-1444AUAUUCUGAAAACCGUAAAGGAA 448 1427 1427-1445 UAUUCUGAAAACCGUAAAGGAAA 4491430 1430-1448 UCUGAAAACCGUAAAGGAAAUCA 450 1431 1431-1449CUGAAAACCGUAAAGGAAAUCAC 451

TABLE 5 EGFR siRNA Sequences Sequence SEQ SEQ position in sense strandsequence ID antisense strand sequence ID hs Id # NM_005228.3 (5′-3′) NO:(5′-3′) NO: 68 68-86 CGGCCGGAGUCCCGAG 452 UAGCUCGGGACUCCGGC 453 CUATTCGTT 71 71-89 CCGGAGUCCCGAGCUA 454 GGCUAGCUCGGGACUCC 455 GCCTT GGTT 7272-90 CGGAGUCCCGAGCUAG 456 GGGCUAGCUCGGGACUC 457 CCCTT CGTT 73 73-91GGAGUCCCGAGCUAGC 458 GGGGCUAGCUCGGGACU 459 CCCTT CCTT 74 74-92GAGUCCCGAGCUAGCC 460 CGGGGCUAGCUCGGGAC 461 CCGTT UCTT 75 75-93AGUCCCGAGCUAGCCC 462 CCGGGGCUAGCUCGGGA 463 CGGTT CUTT 76 76-94GUCCCGAGCUAGCCCC 464 GCCGGGGCUAGCUCGGG 465 GGCTT ACTT 78 78-96CCCGAGCUAGCCCCGG 466 CCGCCGGGGCUAGCUCG 467 CGGTT GGTT 114 114-132GGACGACAGGCCACCU 468 ACGAGGUGGCCUGUCGU 469 CGUTT CCTT 115 115-133GACGACAGGCCACCUC 470 GACGAGGUGGCCUGUCG 471 GUCTT UCTT 116 116-134ACGACAGGCCACCUCG 472 CGACGAGGUGGCCUGUC 473 UCGTT GUTT 117 117-135CGACAGGCCACCUCGU 474 CCGACGAGGUGGCCUGU 475 CGGTT CGTT 118 118-136GACAGGCCACCUCGUC 476 GCCGACGAGGUGGCCUG 477 GGCTT UCTT 120 120-138CAGGCCACCUCGUCGG 478 ACGCCGACGAGGUGGCC 479 CGUTT UGTT 121 121-139AGGCCACCUCGUCGGC 480 GACGCCGACGAGGUGGC 481 GUCTT CUTT 122 122-140GGCCACCUCGUCGGCG 482 GGACGCCGACGAGGUGG 483 UCCTT CCTT 123 123-141GCCACCUCGUCGGCGU 484 CGGACGCCGACGAGGUG 485 CCGTT GCTT 124 124-142CCACCUCGUCGGCGUC 486 GCGGACGCCGACGAGGU 487 CGCTT GGTT 125 125-143CACCUCGUCGGCGUCC 488 GGCGGACGCCGACGAGG 489 GCCTT UGTT 126 126-144ACCUCGUCGGCGUCCG 490 GGGCGGACGCCGACGAG 491 CCCTT GUTT 127 127-145CCUCGUCGGCGUCCGC 492 CGGGCGGACGCCGACGA 493 CCGTT GGTT 128 128-146CUCGUCGGCGUCCGCC 494 UCGGGCGGACGCCGACG 495 CGATT AGTT 129 129-147UCGUCGGCGUCCGCCC 496 CUCGGGCGGACGCCGAC 497 GAGTT GATT 130 130-148CGUCGGCGUCCGCCCG 498 ACUCGGGCGGACGCCGA 499 AGUTT CGTT 131 131-149GUCGGCGUCCGCCCGA 500 GACUCGGGCGGACGCCG 501 GUCTT ACTT 132 132-150UCGGCGUCCGCCCGAG 502 GGACUCGGGCGGACGCC 503 UCCTT GATT 135 135-153GCGUCCGCCCGAGUCC 504 CGGGGACUCGGGCGGAC 505 CCGTT GCTT 136 136-154CGUCCGCCCGAGUCCC 506 GCGGGGACUCGGGCGGA 507 CGCTT CGTT 141 141-159GCCCGAGUCCCCGCCU 508 GCGAGGCGGGGACUCGG 509 CGCTT GCTT 164 164-182AACGCCACAACCACCG 510 GCGCGGUGGUUGUGGCG 511 CGCTT UUTT 165 165-183ACGCCACAACCACCGC 512 UGCGCGGUGGUUGUGGC 513 GCATT GUTT 166 166-184CGCCACAACCACCGCG 514 GUGCGCGGUGGUUGUGG 515 CACTT CGTT 168 168-186CCACAACCACCGCGCA 516 CCGUGCGCGGUGGUUGU 517 CGGTT GGTT 169 169-187CACAACCACCGCGCAC 518 GCCGUGCGCGGUGGUUG 519 GGCTT UGTT 170 170-188ACAACCACCGCGCACG 520 GGCCGUGCGCGGUGGUU 521 GCCTT GUTT 247 247-265AUGCGACCCUCCGGGA 522 CCGUCCCGGAGGGUCGC 523 CGGTT AUTT 248 248-266UGCGACCCUCCGGGAC 524 GCCGUCCCGGAGGGUCG 525 GGCTT CATT 249 249-267GCGACCCUCCGGGACG 526 GGCCGUCCCGGAGGGUC 527 GCCTT GCTT 251 251-269GACCCUCCGGGACGGC 528 CCGGCCGUCCCGGAGGG 529 CGGTT UCTT 252 252-270ACCCUCCGGGACGGCC 530 CCCGGCCGUCCCGGAGG 531 GGGTT GUTT 254 254-272CCUCCGGGACGGCCGG 532 GCCCCGGCCGUCCCGGA 533 GGCTT GGTT 329 329-347AGAAAGUUUGCCAAGG 534 GUGCCUUGGCAAACUUU 535 CACTT CUTT 330 330-348GAAAGUUUGCCAAGGC 536 CGUGCCUUGGCAAACUU 537 ACGTT UCTT 332 332-350AAGUUUGCCAAGGCAC 538 CUCGUGCCUUGGCAAAC 539 GAGTT UUTT 333 333-351AGUUUGCCAAGGCACG 540 ACUCGUGCCUUGGCAAA 541 AGUTT CUTT 334 334-352GUUUGCCAAGGCACGA 542 UACUCGUGCCUUGGCAA 543 GUATT ACTT 335 335-353UUUGCCAAGGCACGAG 544 UUACUCGUGCCUUGGCA 545 UAATT AATT 336 336-354UUGCCAAGGCACGAGU 546 GUUACUCGUGCCUUGGC 547 AACTT AATT 337 337-355UGCCAAGGCACGAGUA 548 UGUUACUCGUGCCUUGG 549 ACATT CATT 338 338-356GCCAAGGCACGAGUAA 550 UUGUUACUCGUGCCUUG 551 CAATT GCTT 361 361-379ACGCAGUUGGGCACUU 552 CAAAAGUGCCCAACUGC 553 UUGTT GUTT 362 362-380CGCAGUUGGGCACUUU 554 UCAAAAGUGCCCAACUG 555 UGATT CGTT 363 363-381GCAGUUGGGCACUUUU 556 UUCAAAAGUGCCCAACU 557 GAATT GCTT 364 364-382CAGUUGGGCACUUUUG 558 CUUCAAAAGUGCCCAAC 559 AAGTT UGTT 365 365-383AGUUGGGCACUUUUGA 560 UCUUCAAAAGUGCCCAA 561 AGATT CUTT 366 366-384GUUGGGCACUUUUGAA 562 AUCUUCAAAAGUGCCCA 563 GAUTT ACTT 367 367-385UUGGGCACUUUUGAAG 564 GAUCUUCAAAAGUGCCC 565 AUCTT AATT 368 368-386UGGGCACUUUUGAAGA 566 UGAUCUUCAAAAGUGCC 567 UCATT CATT 369 369-387GGGCACUUUUGAAGAU 568 AUGAUCUUCAAAAGUGC 569 CAUTT CCTT 377 377-395UUGAAGAUCAUUUUCU 570 CUGAGAAAAUGAUCUUC 571 CAGTT AATT 379 379-397GAAGAUCAUUUUCUCA 572 GGCUGAGAAAAUGAUCU 573 GCCTT UCTT 380 380-398AAGAUCAUUUUCUCAG 574 AGGCUGAGAAAAUGAUC 575 CCUTT UUTT 385 385-403CAUUUUCUCAGCCUCC 576 UCUGGAGGCUGAGAAAA 577 AGATT UGTT 394 394-412AGCCUCCAGAGGAUGU 578 UGAACAUCCUCUGGAGG 579 UCATT CUTT 396 396-414CCUCCAGAGGAUGUUC 580 AUUGAACAUCCUCUGGA 581 AAUTT GGTT 397 397-415CUCCAGAGGAUGUUCA 582 UAUUGAACAUCCUCUGG 583 AUATT AGTT 401 401-419AGAGGAUGUUCAAUAA 584 CAGUUAUUGAACAUCCU 585 CUGTT CUTT 403 403-421AGGAUGUUCAAUAACU 586 CACAGUUAUUGAACAUC 587 GUGTT CUTT 407 407-425UGUUCAAUAACUGUGA 588 ACCUCACAGUUAUUGAA 589 GGUTT CATT 409 409-427UUCAAUAACUGUGAGG 590 CCACCUCACAGUUAUUG 591 UGGTT AATT 410 410-428UCAAUAACUGUGAGGU 592 ACCACCUCACAGUUAUU 593 GGUTT GATT 411 411-429CAAUAACUGUGAGGUG 594 GACCACCUCACAGUUAU 595 GUCTT UGTT 412 412-430AAUAACUGUGAGGUGG 596 GGACCACCUCACAGUUA 597 UCCTT UUTT 413 413-431AUAACUGUGAGGUGGU 598 AGGACCACCUCACAGUU 599 CCUTT AUTT 414 414-432UAACUGUGAGGUGGUC 600 AAGGACCACCUCACAGU 601 CUUTT UATT 416 416-434ACUGUGAGGUGGUCCU 602 CCAAGGACCACCUCACA 603 UGGTT GUTT 418 418-436UGUGAGGUGGUCCUUG 604 UCCCAAGGACCACCUCA 605 GGATT CATT 419 419-437GUGAGGUGGUCCUUGG 606 UUCCCAAGGACCACCUC 607 GAATT ACTT 425 425-443UGGUCCUUGGGAAUUU 608 UCCAAAUUCCCAAGGAC 609 GGATT CATT 431 431-449UUGGGAAUUUGGAAAU 610 GUAAUUUCCAAAUUCCC 611 UACTT AATT 432 432-450UGGGAAUUUGGAAAUU 612 GGUAAUUUCCAAAUUCC 613 ACCTT CATT 433 433-451GGGAAUUUGGAAAUUA 614 AGGUAAUUUCCAAAUUC 615 CCUTT CCTT 434 434-452GGAAUUUGGAAAUUAC 616 UAGGUAAUUUCCAAAUU 617 CUATT CCTT 458 458-476AGAGGAAUUAUGAUCU 618 GAAAGAUCAUAAUUCCU 619 UUCTT CUTT 459 459-477GAGGAAUUAUGAUCUU 620 GGAAAGAUCAUAAUUCC 621 UCCTT UCTT 463 463-481AAUUAUGAUCUUUCCU 622 AGAAGGAAAGAUCAUAA 623 UCUTT UUTT 464 464-482AUUAUGAUCUUUCCUU 624 AAGAAGGAAAGAUCAUA 625 CUUTT AUTT 466 466-484UAUGAUCUUUCCUUCU 626 UUAAGAAGGAAAGAUCA 627 UAATT UATT 468 468-486UGAUCUUUCCUUCUUA 628 CUUUAAGAAGGAAAGAU 629 AAGTT CATT 471 471-489UCUUUCCUUCUUAAAG 630 GGUCUUUAAGAAGGAAA 631 ACCTT GATT 476 476-494CCUUCUUAAAGACCAU 632 UGGAUGGUCUUUAAGAA 633 CCATT GGTT 477 477-495CUUCUUAAAGACCAUC 634 CUGGAUGGUCUUUAAGA 635 CAGTT AGTT 479 479-497UCUUAAAGACCAUCCA 636 UCCUGGAUGGUCUUUAA 637 GGATT GATT 481 481-499UUAAAGACCAUCCAGG 638 CCUCCUGGAUGGUCUUU 639 AGGTT AATT 482 482-500UAAAGACCAUCCAGGA 640 ACCUCCUGGAUGGUCUU 641 GGUTT UATT 492 492-510CCAGGAGGUGGCUGGU 642 AUAACCAGCCACCUCCU 643 UAUTT GGTT 493 493-511CAGGAGGUGGCUGGUU 644 CAUAACCAGCCACCUCC 645 AUGTT UGTT 494 494-512AGGAGGUGGCUGGUUA 646 ACAUAACCAGCCACCUC 647 UGUTT CUTT 495 495-513GGAGGUGGCUGGUUAU 648 GACAUAACCAGCCACCU 649 GUCTT CCTT 496 496-514GAGGUGGCUGGUUAUG 650 GGACAUAACCAGCCACC 651 UCCTT UCTT 497 497-515AGGUGGCUGGUUAUGU 652 AGGACAUAACCAGCCAC 653 CCUTT CUTT 499 499-517GUGGCUGGUUAUGUCC 654 UGAGGACAUAACCAGCC 655 UCATT ACTT 520 520-538GCCCUCAACACAGUGG 656 GCUCCACUGUGUUGAGG 657 AGCTT GCTT 542 542-560UUCCUUUGGAAAACCU 658 UGCAGGUUUUCCAAAGG 659 GCATT AATT 543 543-561UCCUUUGGAAAACCUG 660 CUGCAGGUUUUCCAAAG 661 CAGTT GATT 550 550-568GAAAACCUGCAGAUCA 662 UGAUGAUCUGCAGGUUU 663 UCATT UCTT 551 551-569AAAACCUGCAGAUCAU 664 CUGAUGAUCUGCAGGUU 665 CAGTT UUTT 553 553-571AACCUGCAGAUCAUCA 666 CUCUGAUGAUCUGCAGG 667 GAGTT UUTT 556 556-574CUGCAGAUCAUCAGAG 668 UUCCUCUGAUGAUCUGC 669 GAATT AGTT 586 586-604GAAAAUUCCUAUGCCU 670 CUAAGGCAUAGGAAUUU 671 UAGTT UCTT 587 587-605AAAAUUCCUAUGCCUU 672 GCUAAGGCAUAGGAAUU 673 AGCTT UUTT 589 589-607AAUUCCUAUGCCUUAG 674 CUGCUAAGGCAUAGGAA 675 CAGTT UUTT 592 592-610UCCUAUGCCUUAGCAG 676 AGACUGCUAAGGCAUAG 677 UCUTT GATT 593 593-611CCUAUGCCUUAGCAGU 678 AAGACUGCUAAGGCAUA 679 CUUTT GGTT 594 594-612CUAUGCCUUAGCAGUC 680 UAAGACUGCUAAGGCAU 681 UUATT AGTT 596 596-614AUGCCUUAGCAGUCUU 682 GAUAAGACUGCUAAGGC 683 AUCTT AUTT 597 597-615UGCCUUAGCAGUCUUA 684 AGAUAAGACUGCUAAGG 685 UCUTT CATT 598 598-616GCCUUAGCAGUCUUAU 686 UAGAUAAGACUGCUAAG 687 CUATT GCTT 599 599-617CCUUAGCAGUCUUAUC 688 UUAGAUAAGACUGCUAA 689 UAATT GGTT 600 600-618CUUAGCAGUCUUAUCU 690 GUUAGAUAAGACUGCUA 691 AACTT AGTT 601 601-619UUAGCAGUCUUAUCUA 692 AGUUAGAUAAGACUGCU 693 ACUTT AATT 602 602-620UAGCAGUCUUAUCUAA 694 UAGUUAGAUAAGACUGC 695 CUATT UATT 603 603-621AGCAGUCUUAUCUAAC 696 AUAGUUAGAUAAGACUG 697 UAUTT CUTT 604 604-622GCAGUCUUAUCUAACU 698 CAUAGUUAGAUAAGACU 699 AUGTT GCTT 605 605-623CAGUCUUAUCUAACUA 700 UCAUAGUUAGAUAAGAC 701 UGATT UGTT 608 608-626UCUUAUCUAACUAUGA 702 GCAUCAUAGUUAGAUAA 703 UGCTT GATT 609 609-627CUUAUCUAACUAUGAU 704 UGCAUCAUAGUUAGAUA 705 GCATT AGTT 610 610-628UUAUCUAACUAUGAUG 706 UUGCAUCAUAGUUAGAU 707 CAATT AATT 611 611-629UAUCUAACUAUGAUGC 708 UUUGCAUCAUAGUUAGA 709 AAATT UATT 612 612-630AUCUAACUAUGAUGCA 710 AUUUGCAUCAUAGUUAG 711 AAUTT AUTT 613 613-631UCUAACUAUGAUGCAA 712 UAUUUGCAUCAUAGUUA 713 AUATT GATT 614 614-632CUAACUAUGAUGCAAA 714 UUAUUUGCAUCAUAGUU 715 UAATT AGTT 616 616-634AACUAUGAUGCAAAUA 716 UUUUAUUUGCAUCAUAG 717 AAATT UUTT 622 622-640GAUGCAAAUAAAACCG 718 GUCCGGUUUUAUUUGCA 719 GACTT UCTT 623 623-641AUGCAAAUAAAACCGG 720 AGUCCGGUUUUAUUUGC 721 ACUTT AUTT 624 624-642UGCAAAUAAAACCGGA 722 CAGUCCGGUUUUAUUUG 723 CUGTT CATT 626 626-644CAAAUAAAACCGGACU 724 UUCAGUCCGGUUUUAUU 725 GAATT UGTT 627 627-645AAAUAAAACCGGACUG 726 CUUCAGUCCGGUUUUAU 727 AAGTT UUTT 628 628-646AAUAAAACCGGACUGA 728 CCUUCAGUCCGGUUUUA 729 AGGTT UUTT 630 630-648UAAAACCGGACUGAAG 730 CUCCUUCAGUCCGGUUU 731 GAGTT UATT 631 631-649AAAACCGGACUGAAGG 732 GCUCCUUCAGUCCGGUU 733 AGCTT UUTT 632 632-650AAACCGGACUGAAGGA 734 AGCUCCUUCAGUCCGGU 735 GCUTT UUTT 633 633-651AACCGGACUGAAGGAG 736 CAGCUCCUUCAGUCCGG 737 CUGTT UUTT 644 644-662AGGAGCUGCCCAUGAG 738 UUUCUCAUGGGCAGCUC 739 AAATT CUTT 665 665-683UACAGGAAAUCCUGCA 740 CCAUGCAGGAUUUCCUG 741 UGGTT UATT 668 668-686AGGAAAUCCUGCAUGG 742 GCGCCAUGCAGGAUUUC 743 CGCTT CUTT 669 669-687GGAAAUCCUGCAUGGC 744 GGCGCCAUGCAGGAUUU 745 GCCTT CCTT 670 670-688GAAAUCCUGCAUGGCG 746 CGGCGCCAUGCAGGAUU 747 CCGTT UCTT 671 671-689AAAUCCUGCAUGGCGC 748 ACGGCGCCAUGCAGGAU 749 CGUTT UUTT 672 672-690AAUCCUGCAUGGCGCC 750 CACGGCGCCAUGCAGGA 751 GUGTT UUTT 674 674-692UCCUGCAUGGCGCCGU 752 CGCACGGCGCCAUGCAG 753 GCGTT GATT 676 676-694CUGCAUGGCGCCGUGC 754 ACCGCACGGCGCCAUGC 755 GGUTT AGTT 677 677-695UGCAUGGCGCCGUGCG 756 AACCGCACGGCGCCAUG 757 GUUTT CATT 678 678-696GCAUGGCGCCGUGCGG 758 GAACCGCACGGCGCCAU 759 UUCTT GCTT 680 680-698AUGGCGCCGUGCGGUU 760 CUGAACCGCACGGCGCC 761 CAGTT AUTT 681 681-699UGGCGCCGUGCGGUUC 762 GCUGAACCGCACGGCGC 763 AGCTT CATT 682 682-700GGCGCCGUGCGGUUCA 764 UGCUGAACCGCACGGCG 765 GCATT CCTT 683 683-701GCGCCGUGCGGUUCAG 766 UUGCUGAACCGCACGGC 767 CAATT GCTT 684 684-702CGCCGUGCGGUUCAGC 768 GUUGCUGAACCGCACGG 769 AACTT CGTT 685 685-703GCCGUGCGGUUCAGCA 770 UGUUGCUGAACCGCACG 771 ACATT GCTT 686 686-704CCGUGCGGUUCAGCAA 772 UUGUUGCUGAACCGCAC 773 CAATT GGTT 688 688-706GUGCGGUUCAGCAACA 774 GGUUGUUGCUGAACCGC 775 ACCTT ACTT 690 690-708GCGGUUCAGCAACAAC 776 AGGGUUGUUGCUGAACC 777 CCUTT GCTT 692 692-710GGUUCAGCAACAACCC 778 GCAGGGUUGUUGCUGAA 779 UGCTT CCTT 698 698-716GCAACAACCCUGCCCU 780 CACAGGGCAGGGUUGUU 781 GUGTT GCTT 700 700-718AACAACCCUGCCCUGU 782 UGCACAGGGCAGGGUUG 783 GCATT UUTT 719 719-737ACGUGGAGAGCAUCCA 784 CACUGGAUGCUCUCCAC 785 GUGTT GUTT 720 720-738CGUGGAGAGCAUCCAG 786 CCACUGGAUGCUCUCCA 787 UGGTT CGTT 721 721-739GUGGAGAGCAUCCAGU 788 GCCACUGGAUGCUCUCC 789 GGCTT ACTT 724 724-742GAGAGCAUCCAGUGGC 790 CCCGCCACUGGAUGCUC 791 GGGTT UCTT 725 725-743AGAGCAUCCAGUGGCG 792 UCCCGCCACUGGAUGCU 793 GGATT CUTT 726 726-744GAGCAUCCAGUGGCGG 794 GUCCCGCCACUGGAUGC 795 GACTT UCTT 733 733-751CAGUGGCGGGACAUAG 796 UGACUAUGUCCCGCCAC 797 UCATT UGTT 734 734-752AGUGGCGGGACAUAGU 798 CUGACUAUGUCCCGCCA 799 CAGTT CUTT 736 736-754UGGCGGGACAUAGUCA 800 UGCUGACUAUGUCCCGC 801 GCATT CATT 737 737-755GGCGGGACAUAGUCAG 802 CUGCUGACUAUGUCCCG 803 CAGTT CCTT 763 763-781CUCAGCAACAUGUCGA 804 CCAUCGACAUGUUGCUG 805 UGGTT AGTT 765 765-783CAGCAACAUGUCGAUG 806 GUCCAUCGACAUGUUGC 807 GACTT UGTT 766 766-784AGCAACAUGUCGAUGG 808 AGUCCAUCGACAUGUUG 809 ACUTT CUTT 767 767-785GCAACAUGUCGAUGGA 810 AAGUCCAUCGACAUGUU 811 CUUTT GCTT 769 769-787AACAUGUCGAUGGACU 812 GGAAGUCCAUCGACAUG 813 UCCTT UUTT 770 770-788ACAUGUCGAUGGACUU 814 UGGAAGUCCAUCGACAU 815 CCATT GUTT 771 771-789CAUGUCGAUGGACUUC 816 CUGGAAGUCCAUCGACA 817 CAGTT UGTT 772 772-790AUGUCGAUGGACUUCC 818 UCUGGAAGUCCAUCGAC 819 AGATT AUTT 775 775-793UCGAUGGACUUCCAGA 820 GGUUCUGGAAGUCCAUC 821 ACCTT GATT 789 789-807GAACCACCUGGGCAGC 822 GCAGCUGCCCAGGUGGU 823 UGCTT UCTT 798 798-816GGGCAGCUGCCAAAAG 824 ACACUUUUGGCAGCUGC 825 UGUTT CCTT 800 800-818GCAGCUGCCAAAAGUG 826 UCACACUUUUGGCAGCU 827 UGATT GCTT 805 805-823UGCCAAAAGUGUGAUC 828 UUGGAUCACACUUUUGG 829 CAATT CATT 806 806-824GCCAAAAGUGUGAUCC 830 CUUGGAUCACACUUUUG 831 AAGTT GCTT 807 807-825CCAAAAGUGUGAUCCA 832 GCUUGGAUCACACUUUU 833 AGCTT GGTT 810 810-828AAAGUGUGAUCCAAGC 834 ACAGCUUGGAUCACACU 835 UGUTT UUTT 814 814-832UGUGAUCCAAGCUGUC 836 UGGGACAGCUUGGAUCA 837 CCATT CATT 815 815-833GUGAUCCAAGCUGUCC 838 UUGGGACAGCUUGGAUC 839 CAATT ACTT 817 817-835GAUCCAAGCUGUCCCA 840 CAUUGGGACAGCUUGGA 841 AUGTT UCTT 818 818-836AUCCAAGCUGUCCCAA 842 CCAUUGGGACAGCUUGG 843 UGGTT AUTT 819 819-837UCCAAGCUGUCCCAAU 844 CCCAUUGGGACAGCUUG 845 GGGTT GATT 820 820-838CCAAGCUGUCCCAAUG 846 UCCCAUUGGGACAGCUU 847 GGATT GGTT 821 821-839CAAGCUGUCCCAAUGG 848 CUCCCAUUGGGACAGCU 849 GAGTT UGTT 823 823-841AGCUGUCCCAAUGGGA 850 AGCUCCCAUUGGGACAG 851 GCUTT CUTT 826 826-844UGUCCCAAUGGGAGCU 852 AGCAGCUCCCAUUGGGA 853 GCUTT CATT 847 847-865GGUGCAGGAGAGGAGA 854 AGUUCUCCUCUCCUGCA 855 ACUTT CCTT 871 871-889AAACUGACCAAAAUCA 856 AGAUGAUUUUGGUCAGU 857 UCUTT UUTT 872 872-890AACUGACCAAAAUCAU 858 CAGAUGAUUUUGGUCAG 859 CUGTT UUTT 873 873-891ACUGACCAAAAUCAUC 860 ACAGAUGAUUUUGGUCA 861 UGUTT GUTT 877 877-895ACCAAAAUCAUCUGUG 862 GGGCACAGAUGAUUUUG 863 CCCTT GUTT 878 878-896CCAAAAUCAUCUGUGC 864 UGGGCACAGAUGAUUUU 865 CCATT GGTT 881 881-899AAAUCAUCUGUGCCCA 866 UGCUGGGCACAGAUGAU 867 GCATT UUTT 890 890-908GUGCCCAGCAGUGCUC 868 CCGGAGCACUGCUGGGC 869 CGGTT ACTT 892 892-910GCCCAGCAGUGCUCCG 870 GCCCGGAGCACUGCUGG 871 GGCTT GCTT 929 929-947CCAGUGACUGCUGCCA 872 UUGUGGCAGCAGUCACU 873 CAATT GGTT 930 930-948CAGUGACUGCUGCCAC 874 GUUGUGGCAGCAGUCAC 875 AACTT UGTT 979 979-997GAGAGCGACUGCCUGG 876 AGACCAGGCAGUCGCUC 877 UCUTT UCTT 980 980-998AGAGCGACUGCCUGGU 878 CAGACCAGGCAGUCGCU 879 CUGTT CUTT 981 981-999GAGCGACUGCCUGGUC 880 GCAGACCAGGCAGUCGC 881 UGCTT UCTT 982  982-1000AGCGACUGCCUGGUCU 882 GGCAGACCAGGCAGUCG 883 GCCTT CUTT 983  983-1001GCGACUGCCUGGUCUG 884 CGGCAGACCAGGCAGUC 885 CCGTT GCTT 984  984-1002CGACUGCCUGGUCUGC 886 GCGGCAGACCAGGCAGU 887 CGCTT CGTT 989  989-1007GCCUGGUCUGCCGCAA 888 AAUUUGCGGCAGACCAG 889 AUUTT GCTT 990  990-1008CCUGGUCUGCCGCAAA 890 GAAUUUGCGGCAGACCA 891 UUCTT GGTT 991  991-1009CUGGUCUGCCGCAAAU 892 GGAAUUUGCGGCAGACC 893 UCCTT AGTT 992  992-1010UGGUCUGCCGCAAAUU 894 CGGAAUUUGCGGCAGAC 895 CCGTT CATT 994  994-1012GUCUGCCGCAAAUUCC 896 CUCGGAAUUUGCGGCAG 897 GAGTT ACTT 995  995-1013UCUGCCGCAAAUUCCG 898 UCUCGGAAUUUGCGGCA 899 AGATT GATT 996  996-1014CUGCCGCAAAUUCCGA 900 GUCUCGGAAUUUGCGGC 901 GACTT AGTT 997  997-1015UGCCGCAAAUUCCGAG 902 CGUCUCGGAAUUUGCGG 903 ACGTT CATT 999  999-1017CCGCAAAUUCCGAGAC 904 UUCGUCUCGGAAUUUGC 905 GAATT GGTT 1004 1004-1022AAUUCCGAGACGAAGC 906 GUGGCUUCGUCUCGGAA 907 CACTT UUTT 1005 1005-1023AUUCCGAGACGAAGCC 908 CGUGGCUUCGUCUCGGA 909 ACGTT AUTT 1006 1006-1024UUCCGAGACGAAGCCA 910 ACGUGGCUUCGUCUCGG 911 CGUTT AATT 1007 1007-1025UCCGAGACGAAGCCAC 912 CACGUGGCUUCGUCUCG 913 GUGTT GATT 1008 1008-1026CCGAGACGAAGCCACG 914 GCACGUGGCUUCGUCUC 915 UGCTT GGTT 1010 1010-1028GAGACGAAGCCACGUG 916 UUGCACGUGGCUUCGUC 917 CAATT UCTT 1013 1013-1031ACGAAGCCACGUGCAA 918 UCCUUGCACGUGGCUUC 919 GGATT GUTT 1014 1014-1032CGAAGCCACGUGCAAG 920 GUCCUUGCACGUGGCUU 921 GACTT CGTT 1015 1015-1033GAAGCCACGUGCAAGG 922 UGUCCUUGCACGUGGCU 923 ACATT UCTT 1016 1016-1034AAGCCACGUGCAAGGA 924 GUGUCCUUGCACGUGGC 925 CACTT UUTT 1040 1040-1058CCCCACUCAUGCUCUA 926 UUGUAGAGCAUGAGUGG 927 CAATT GGTT 1042 1042-1060CCACUCAUGCUCUACA 928 GGUUGUAGAGCAUGAGU 929 ACCTT GGTT 1044 1044-1062ACUCAUGCUCUACAAC 930 GGGGUUGUAGAGCAUGA 931 CCCTT GUTT 1047 1047-1065CAUGCUCUACAACCCC 932 GGUGGGGUUGUAGAGCA 933 ACCTT UGTT 1071 1071-1089CCAGAUGGAUGUGAAC 934 GGGGUUCACAUCCAUCU 935 CCCTT GGTT 1073 1073-1091AGAUGGAUGUGAACCC 936 UCGGGGUUCACAUCCAU 937 CGATT CUTT 1074 1074-1092GAUGGAUGUGAACCCC 938 CUCGGGGUUCACAUCCA 939 GAGTT UCTT 1075 1075-1093AUGGAUGUGAACCCCG 940 CCUCGGGGUUCACAUCC 941 AGGTT AUTT 1077 1077-1095GGAUGUGAACCCCGAG 942 GCCCUCGGGGUUCACAU 943 GGCTT CCTT 1078 1078-1096GAUGUGAACCCCGAGG 944 UGCCCUCGGGGUUCACA 945 GCATT UCTT 1080 1080-1098UGUGAACCCCGAGGGC 946 UUUGCCCUCGGGGUUCA 947 AAATT CATT 1084 1084-1102AACCCCGAGGGCAAAU 948 UGUAUUUGCCCUCGGGG 949 ACATT UUTT 1085 1085-1103ACCCCGAGGGCAAAUA 950 CUGUAUUUGCCCUCGGG 951 CAGTT GUTT 1087 1087-1105CCCGAGGGCAAAUACA 952 AGCUGUAUUUGCCCUCG 953 GCUTT GGTT 1088 1088-1106CCGAGGGCAAAUACAG 954 AAGCUGUAUUUGCCCUC 955 CUUTT GGTT 1089 1089-1107CGAGGGCAAAUACAGC 956 AAAGCUGUAUUUGCCCU 957 UUUTT CGTT 1096 1096-1114AAAUACAGCUUUGGUG 958 UGGCACCAAAGCUGUAU 959 CCATT UUTT 1097 1097-1115AAUACAGCUUUGGUGC 960 GUGGCACCAAAGCUGUA 961 CACTT UUTT 1098 1098-1116AUACAGCUUUGGUGCC 962 GGUGGCACCAAAGCUGU 963 ACCTT AUTT 1104 1104-1122CUUUGGUGCCACCUGC 964 CACGCAGGUGGCACCAA 965 GUGTT AGTT 1106 1106-1124UUGGUGCCACCUGCGU 966 UUCACGCAGGUGGCACC 967 GAATT AATT 1112 1112-1130CCACCUGCGUGAAGAA 968 CACUUCUUCACGCAGGU 969 GUGTT GGTT 1116 1116-1134CUGCGUGAAGAAGUGU 970 GGGACACUUCUUCACGC 971 CCCTT AGTT 1117 1117-1135UGCGUGAAGAAGUGUC 972 GGGGACACUUCUUCACG 973 CCCTT CATT 1118 1118-1136GCGUGAAGAAGUGUCC 974 CGGGGACACUUCUUCAC 975 CCGTT GCTT 1119 1119-1137CGUGAAGAAGUGUCCC 976 ACGGGGACACUUCUUCA 977 CGUTT CGTT 1120 1120-1138GUGAAGAAGUGUCCCC 978 UACGGGGACACUUCUUC 979 GUATT ACTT 1121 1121-1139UGAAGAAGUGUCCCCG 980 UUACGGGGACACUUCUU 981 UAATT CATT 1122 1122-1140GAAGAAGUGUCCCCGU 982 AUUACGGGGACACUUCU 983 AAUTT UCTT 1123 1123-1141AAGAAGUGUCCCCGUA 984 AAUUACGGGGACACUUC 985 AUUTT UUTT 1124 1124-1142AGAAGUGUCCCCGUAA 986 UAAUUACGGGGACACUU 987 UUATT CUTT 1125 1125-1143GAAGUGUCCCCGUAAU 988 AUAAUUACGGGGACACU 989 UAUTT UCTT 1126 1126-1144AAGUGUCCCCGUAAUU 990 CAUAAUUACGGGGACAC 991 AUGTT UUTT 1127 1127-1145AGUGUCCCCGUAAUUA 992 ACAUAAUUACGGGGACA 993 UGUTT CUTT 1128 1128-1146GUGUCCCCGUAAUUAU 994 CACAUAAUUACGGGGAC 995 GUGTT ACTT 1129 1129-1147UGUCCCCGUAAUUAUG 996 CCACAUAAUUACGGGGA 997 UGGTT CATT 1130 1130-1148GUCCCCGUAAUUAUGU 998 ACCACAUAAUUACGGGG 999 GGUTT ACTT 1132 1132-1150CCCCGUAAUUAUGUGG 1000 UCACCACAUAAUUACGG 1001 UGATT GGTT 1134 1134-1152CCGUAAUUAUGUGGUG 1002 UGUCACCACAUAAUUAC 1003 ACATT GGTT 1136 1136-1154GUAAUUAUGUGGUGAC 1004 UCUGUCACCACAUAAUU 1005 AGATT ACTT 1137 1137-1155UAAUUAUGUGGUGACA 1006 AUCUGUCACCACAUAAU 1007 GAUTT UATT 1138 1138-1156AAUUAUGUGGUGACAG 1008 GAUCUGUCACCACAUAA 1009 AUCTT UUTT 1139 1139-1157AUUAUGUGGUGACAGA 1010 UGAUCUGUCACCACAUA 1011 UCATT AUTT 1140 1140-1158UUAUGUGGUGACAGAU 1012 GUGAUCUGUCACCACAU 1013 CACTT AATT 1142 1142-1160AUGUGGUGACAGAUCA 1014 CCGUGAUCUGUCACCAC 1015 CGGTT AUTT 1145 1145-1163UGGUGACAGAUCACGG 1016 GAGCCGUGAUCUGUCAC 1017 CUCTT CATT 1147 1147-1165GUGACAGAUCACGGCU 1018 ACGAGCCGUGAUCUGUC 1019 CGUTT ACTT 1148 1148-1166UGACAGAUCACGGCUC 1020 CACGAGCCGUGAUCUGU 1021 GUGTT CATT 1149 1149-1167GACAGAUCACGGCUCG 1022 GCACGAGCCGUGAUCUG 1023 UGCTT UCTT 1150 1150-1168ACAGAUCACGGCUCGU 1024 CGCACGAGCCGUGAUCU 1025 GCGTT GUTT 1151 1151-1169CAGAUCACGGCUCGUG 1026 ACGCACGAGCCGUGAUC 1027 CGUTT UGTT 1152 1152-1170AGAUCACGGCUCGUGC 1028 GACGCACGAGCCGUGAU 1029 GUCTT CUTT 1153 1153-1171GAUCACGGCUCGUGCG 1030 GGACGCACGAGCCGUGA 1031 UCCTT UCTT 1154 1154-1172AUCACGGCUCGUGCGU 1032 CGGACGCACGAGCCGUG 1033 CCGTT AUTT 1155 1155-1173UCACGGCUCGUGCGUC 1034 UCGGACGCACGAGCCGU 1035 CGATT GATT 1156 1156-1174CACGGCUCGUGCGUCC 1036 CUCGGACGCACGAGCCG 1037 GAGTT UGTT 1157 1157-1175ACGGCUCGUGCGUCCG 1038 GCUCGGACGCACGAGCC 1039 AGCTT GUTT 1160 1160-1178GCUCGUGCGUCCGAGC 1040 CAGGCUCGGACGCACGA 1041 CUGTT GCTT 1200 1200-1218GGAGGAAGACGGCGUC 1042 GCGGACGCCGUCUUCCU 1043 CGCTT CCTT 1201 1201-1219GAGGAAGACGGCGUCC 1044 UGCGGACGCCGUCUUCC 1045 GCATT UCTT 1203 1203-1221GGAAGACGGCGUCCGC 1046 CUUGCGGACGCCGUCUU 1047 AAGTT CCTT 1204 1204-1222GAAGACGGCGUCCGCA 1048 ACUUGCGGACGCCGUCU 1049 AGUTT UCTT 1205 1205-1223AAGACGGCGUCCGCAA 1050 CACUUGCGGACGCCGUC 1051 GUGTT UUTT 1207 1207-1225GACGGCGUCCGCAAGU 1052 UACACUUGCGGACGCCG 1053 GUATT UCTT 1208 1208-1226ACGGCGUCCGCAAGUG 1054 UUACACUUGCGGACGCC 1055 UAATT GUTT 1211 1211-1229GCGUCCGCAAGUGUAA 1056 UUCUUACACUUGCGGAC 1057 GAATT GCTT 1212 1212-1230CGUCCGCAAGUGUAAG 1058 CUUCUUACACUUGCGGA 1059 AAGTT CGTT 1213 1213-1231GUCCGCAAGUGUAAGA 1060 ACUUCUUACACUUGCGG 1061 AGUTT ACTT 1214 1214-1232UCCGCAAGUGUAAGAA 1062 CACUUCUUACACUUGCG 1063 GUGTT GATT 1215 1215-1233CCGCAAGUGUAAGAAG 1064 GCACUUCUUACACUUGC 1065 UGCTT GGTT 1216 1216-1234CGCAAGUGUAAGAAGU 1066 CGCACUUCUUACACUUG 1067 GCGTT CGTT 1217 1217-1235GCAAGUGUAAGAAGUG 1068 UCGCACUUCUUACACUU 1069 CGATT GCTT 1219 1219-1237AAGUGUAAGAAGUGCG 1070 CUUCGCACUUCUUACAC 1071 AAGTT UUTT 1220 1220-1238AGUGUAAGAAGUGCGA 1072 CCUUCGCACUUCUUACA 1073 AGGTT CUTT 1221 1221-1239GUGUAAGAAGUGCGAA 1074 CCCUUCGCACUUCUUAC 1075 GGGTT ACTT 1222 1222-1240UGUAAGAAGUGCGAAG 1076 GCCCUUCGCACUUCUUA 1077 GGCTT CATT 1223 1223-1241GUAAGAAGUGCGAAGG 1078 GGCCCUUCGCACUUCUU 1079 GCCTT ACTT 1224 1224-1242UAAGAAGUGCGAAGGG 1080 AGGCCCUUCGCACUUCU 1081 CCUTT UATT 1225 1225-1243AAGAAGUGCGAAGGGC 1082 AAGGCCCUUCGCACUUC 1083 CUUTT UUTT 1226 1226-1244AGAAGUGCGAAGGGCC 1084 CAAGGCCCUUCGCACUU 1085 UUGTT CUTT 1229 1229-1247AGUGCGAAGGGCCUUG 1086 CGGCAAGGCCCUUCGCA 1087 CCGTT CUTT 1230 1230-1248GUGCGAAGGGCCUUGC 1088 GCGGCAAGGCCCUUCGC 1089 CGCTT ACTT 1231 1231-1249UGCGAAGGGCCUUGCC 1090 UGCGGCAAGGCCCUUCG 1091 GCATT CATT 1232 1232-1250GCGAAGGGCCUUGCCG 1092 UUGCGGCAAGGCCCUUC 1093 CAATT GCTT 1233 1233-1251CGAAGGGCCUUGCCGC 1094 UUUGCGGCAAGGCCCUU 1095 AAATT CGTT 1235 1235-1253AAGGGCCUUGCCGCAA 1096 ACUUUGCGGCAAGGCCC 1097 AGUTT UUTT 1236 1236-1254AGGGCCUUGCCGCAAA 1098 CACUUUGCGGCAAGGCC 1099 GUGTT CUTT 1237 1237-1255GGGCCUUGCCGCAAAG 1100 ACACUUUGCGGCAAGGC 1101 UGUTT CCTT 1238 1238-1256GGCCUUGCCGCAAAGU 1102 CACACUUUGCGGCAAGG 1103 GUGTT CCTT 1239 1239-1257GCCUUGCCGCAAAGUG 1104 ACACACUUUGCGGCAAG 1105 UGUTT GCTT 1241 1241-1259CUUGCCGCAAAGUGUG 1106 UUACACACUUUGCGGCA 1107 UAATT AGTT 1261 1261-1279GGAAUAGGUAUUGGUG 1108 AUUCACCAAUACCUAUU 1109 AAUTT CCTT 1262 1262-1280GAAUAGGUAUUGGUGA 1110 AAUUCACCAAUACCUAU 1111 AUUTT UCTT 1263 1263-1281AAUAGGUAUUGGUGAA 1112 AAAUUCACCAAUACCUA 1113 UUUTT UUTT 1264 1264-1282AUAGGUAUUGGUGAAU 1114 UAAAUUCACCAAUACCU 1115 UUATT AUTT 1266 1266-1284AGGUAUUGGUGAAUUU 1116 UUUAAAUUCACCAAUAC 1117 AAATT CUTT 1267 1267-1285GGUAUUGGUGAAUUUA 1118 CUUUAAAUUCACCAAUA 1119 AAGTT CCTT 1289 1289-1307CACUCUCCAUAAAUGC 1120 GUAGCAUUUAUGGAGAG 1121 UACTT UGTT 1313 1313-1331UUAAACACUUCAAAAA 1122 CAGUUUUUGAAGUGUUU 1123 CUGTT AATT 1320 1320-1338CUUCAAAAACUGCACC 1124 GGAGGUGCAGUUUUUGA 1125 UCCTT AGTT 1321 1321-1339UUCAAAAACUGCACCU 1126 UGGAGGUGCAGUUUUUG 1127 CCATT AATT 1322 1322-1340UCAAAAACUGCACCUC 1128 AUGGAGGUGCAGUUUUU 1129 CAUTT GATT 1323 1323-1341CAAAAACUGCACCUCC 1130 GAUGGAGGUGCAGUUUU 1131 AUCTT UGTT 1324 1324-1342AAAAACUGCACCUCCA 1132 UGAUGGAGGUGCAGUUU 1133 UCATT UUTT 1328 1328-1346ACUGCACCUCCAUCAG 1134 CCACUGAUGGAGGUGCA 1135 UGGTT GUTT 1332 1332-1350CACCUCCAUCAGUGGC 1136 AUCGCCACUGAUGGAGG 1137 GAUTT UGTT 1333 1333-1351ACCUCCAUCAGUGGCG 1138 GAUCGCCACUGAUGGAG 1139 AUCTT GUTT 1335 1335-1353CUCCAUCAGUGGCGAU 1140 GAGAUCGCCACUGAUGG 1141 CUCTT AGTT 1338 1338-1356CAUCAGUGGCGAUCUC 1142 GUGGAGAUCGCCACUGA 1143 CACTT UGTT 1344 1344-1362UGGCGAUCUCCACAUC 1144 CAGGAUGUGGAGAUCGC 1145 CUGTT CATT 1345 1345-1363GGCGAUCUCCACAUCC 1146 GCAGGAUGUGGAGAUCG 1147 UGCTT CCTT 1346 1346-1364GCGAUCUCCACAUCCU 1148 GGCAGGAUGUGGAGAUC 1149 GCCTT GCTT 1347 1347-1365CGAUCUCCACAUCCUG 1150 CGGCAGGAUGUGGAGAU 1151 CCGTT CGTT 1348 1348-1366GAUCUCCACAUCCUGC 1152 CCGGCAGGAUGUGGAGA 1153 CGGTT UCTT 1353 1353-1371CCACAUCCUGCCGGUG 1154 UGCCACCGGCAGGAUGU 1155 GCATT GGTT 1354 1354-1372CACAUCCUGCCGGUGG 1156 AUGCCACCGGCAGGAUG 1157 CAUTT UGTT 1355 1355-1373ACAUCCUGCCGGUGGC 1158 AAUGCCACCGGCAGGAU 1159 AUUTT GUTT 1357 1357-1375AUCCUGCCGGUGGCAU 1160 UAAAUGCCACCGGCAGG 1161 UUATT AUTT 1360 1360-1378CUGCCGGUGGCAUUUA 1162 CCCUAAAUGCCACCGGC 1163 GGGTT AGTT 1361 1361-1379UGCCGGUGGCAUUUAG 1164 CCCCUAAAUGCCACCGG 1165 GGGTT CATT 1362 1362-1380GCCGGUGGCAUUUAGG 1166 ACCCCUAAAUGCCACCG 1167 GGUTT GCTT 1363 1363-1381CCGGUGGCAUUUAGGG 1168 CACCCCUAAAUGCCACC 1169 GUGTT GGTT 1366 1366-1384GUGGCAUUUAGGGGUG 1170 AGUCACCCCUAAAUGCC 1171 ACUTT ACTT 1369 1369-1387GCAUUUAGGGGUGACU 1172 AGGAGUCACCCCUAAAU 1173 CCUTT GCTT 1370 1370-1388CAUUUAGGGGUGACUC 1174 AAGGAGUCACCCCUAAA 1175 CUUTT UGTT 1371 1371-1389AUUUAGGGGUGACUCC 1176 GAAGGAGUCACCCCUAA 1177 UUCTT AUTT 1372 1372-1390UUUAGGGGUGACUCCU 1178 UGAAGGAGUCACCCCUA 1179 UCATT AATT 1373 1373-1391UUAGGGGUGACUCCUU 1180 GUGAAGGAGUCACCCCU 1181 CACTT AATT 1374 1374-1392UAGGGGUGACUCCUUC 1182 UGUGAAGGAGUCACCCC 1183 ACATT UATT 1404 1404-1422UCUGGAUCCACAGGAA 1184 CAGUUCCUGUGGAUCCA 1185 CUGTT GATT 1408 1408-1426GAUCCACAGGAACUGG 1186 UAUCCAGUUCCUGUGGA 1187 AUATT UCTT 1409 1409-1427AUCCACAGGAACUGGA 1188 AUAUCCAGUUCCUGUGG 1189 UAUTT AUTT 1411 1411-1429CCACAGGAACUGGAUA 1190 GAAUAUCCAGUUCCUGU 1191 UUCTT GGTT 1412 1412-1430CACAGGAACUGGAUAU 1192 AGAAUAUCCAGUUCCUG 1193 UCUTT UGTT 1419 1419-1437ACUGGAUAUUCUGAAA 1194 GGUUUUCAGAAUAUCCA 1195 ACCTT GUTT 1426 1426-1444AUUCUGAAAACCGUAA 1196 CCUUUACGGUUUUCAGA 1197 AGGTT AUTT 1427 1427-1445UUCUGAAAACCGUAAA 1198 UCCUUUACGGUUUUCAG 1199 GGATT AATT 1430 1430-1448UGAAAACCGUAAAGGA 1200 AUUUCCUUUACGGUUUU 1201 AAUTT CATT 1431 1431-1449GAAAACCGUAAAGGAA 1202 GAUUUCCUUUACGGUUU 1203 AUCTT UCTT

TABLE 6 EGFR siRNA Sequences with Chemical Modifications Sequence SEQSEQ position in sense strand sequence ID antisense strand sequence ID hsId # NM_005228.3 (5′-3′) NO: (5′-3′) NO: 68 68-86cgGfcCfgGfaGfuCfcCfgAfg 1204 UfAfgCfuCfgGfgAfcUfcCfgGf 1205 CfuAfdTsdTcCfgdTsdT 71 71-89 ccGfgAfgUfcCfcGfaGfcUfa 1206GfGfcUfaGfcUfcGfgGfaCfuCf 1207 GfcCfdTsdT cGfgdTsdT 72 72-90cgGfaGfuCfcCfgAfgCfuAfg 1208 GfGfgCfuAfgCfuCfgGfgAfcUf 1209 CfcCfdTsdTcCfgdTsdT 73 73-91 ggAfgUfcCfcGfaGfcUfaGfc 1210GfGfgGfcUfaGfcUfcGfgGfaCf 1211 CfcCfdTsdT uCfcdTsdT 74 74-92gaGfuCfcCfgAfgCfuAfgCfc 1212 CfGfgGfgCfuAfgCfuCfgGfgAf 1213 CfcGfdTsdTcUfcdTsdT 75 75-93 agUfcCfcGfaGfcUfaGfcCfcC 1214CfCfgGfgGfcUfaGfcUfcGfgGf 1215 fgGfdTsdT aCfudTsdT 76 76-94guCfcCfgAfgCfuAfgCfcCfc 1216 GfCfcGfgGfgCfuAfgCfuCfgGf 1217 GfgCfdTsdTgAfcdTsdT 78 78-96 ccCfgAfgCfuAfgCfcCfcGfg 1218CfCfgCfcGfgGfgCfuAfgCfuCf 1219 CfgGfdTsdT gGfgdTsdT 114 114-132ggAfcGfaCfaGfgCfcAfcCfu 1220 AfCfgAfgGfuGfgCfcUfgUfcGf 1221 CfgUfdTsdTuCfcdTsdT 115 115-133 gaCfgAfcAfgGfcCfaCfcUfc 1222GfAfcGfaGfgUfgGfcCfuGfuCf 1223 GfuCfdTsdT gUfcdTsdT 116 116-134acGfaCfaGfgCfcAfcCfuCfg 1224 CfGfaCfgAfgGfuGfgCfcUfgUf 1225 UfcGfdTsdTcGfudTsdT 117 117-135 cgAfcAfgGfcCfaCfcUfcGfu 1226CfCfgAfcGfaGfgUfgGfcCfuGf 1227 CfgGfdTsdT uCfgdTsdT 118 118-136gaCfaGfgCfcAfcCfuCfgUfc 1228 GfCfcGfaCfgAfgGfuGfgCfcUf 1229 GfgCfdTsdTgUfcdTsdT 120 120-138 caGfgCfcAfcCfuCfgUfcGfg 1230AfCfgCfcGfaCfgAfgGfuGfgCf 1231 CfgUfdTsdT cUfgdTsdT 121 121-139agGfcCfaCfcUfcGfuCfgGfc 1232 GfAfcGfcCfgAfcGfaGfgUfgGf 1233 GfuCfdTsdTcCfudTsdT 122 122-140 ggCfcAfcCfuCfgUfcGfgCfg 1234GfGfaCfgCfcGfaCfgAfgGfuGf 1235 UfcCfdTsdT gCfcdTsdT 123 123-141gcCfaCfcUfcGfuCfgGfcGfu 1236 CfGfgAfcGfcCfgAfcGfaGfgUf 1237 CfcGfdTsdTgGfcdTsdT 124 124-142 ccAfcCfuCfgUfcGfgCfgUfc 1238GfCfgGfaCfgCfcGfaCfgAfgGf 1239 CfgCfdTsdT uGfgdTsdT 125 125-143caCfcUfcGfuCfgGfcGfuCfc 1240 GfGfcGfgAfcGfcCfgAfcGfaGf 1241 GfcCfdTsdTgUfgdTsdT 126 126-144 acCfuCfgUfcGfgCfgUfcCfg 1242GfGfgCfgGfaCfgCfcGfaCfgAf 1243 CfcCfdTsdT gGfudTsdT 127 127-145ccUfcGfuCfgGfcGfuCfcGfc 1244 CfGfgGfcGfgAfcGfcCfgAfcGf 1245 CfcGfdTsdTaGfgdTsdT 128 128-146 cuCfgUfcGfgCfgUfcCfgCfc 1246UfCfgGfgCfgGfaCfgCfcGfaCf 1247 CfgAfdTsdT gAfgdTsdT 129 129-147ucGfuCfgGfcGfuCfcGfcCfc 1248 CfUfcGfgGfcGfgAfcGfcCfgAf 1249 GfaGfdTsdTcGfadTsdT 130 130-148 cgUfcGfgCfgUfcCfgCfcCfg 1250AfCfuCfgGfgCfgGfaCfgCfcGf 1251 AfgUfdTsdT aCfgdTsdT 131 131-149guCfgGfcGfuCfcGfcCfcGfa 1252 GfAfcUfcGfgGfcGfgAfcGfcCf 1253 GfuCfdTsdTgAfcdTsdT 132 132-150 ucGfgCfgUfcCfgCfcCfgAfg 1254GfGfaCfuCfgGfgCfgGfaCfgCf 1255 UfcCfdTsdT cGfadTsdT 135 135-153gcGfuCfcGfcCfcGfaGfuCfc 1256 CfGfgGfgAfcUfcGfgGfcGfgAf 1257 CfcGfdTsdTcGfcdTsdT 136 136-154 cgUfcCfgCfcCfgAfgUfcCfc 1258GfCfgGfgGfaCfuCfgGfgCfgGf 1259 CfgCfdTsdT aCfgdTsdT 141 141-159gcCfcGfaGfuCfcCfcGfcCfuC 1260 GfCfgAfgGfcGfgGfgAfcUfcGf 1261 fgCfdTsdTgGfcdTsdT 164 164-182 aaCfgCfcAfcAfaCfcAfcCfgC 1262GfCfgCfgGfuGfgUfuGfuGfgC 1263 fgCfdTsdT fgUfudTsdT 165 165-183acGfcCfaCfaAfcCfaCfcGfcG 1264 UfGfcGfcGfgUfgGfuUfgUfgG 1265 fcAfdTsdTfcGfudTsdT 166 166-184 cgCfcAfcAfaCfcAfcCfgCfgC 1266GfUfgCfgCfgGfuGfgUfuGfuG 1267 faCfdTsdT fgCfgdTsdT 168 168-186ccAfcAfaCfcAfcCfgCfgCfaC 1268 CfCfgUfgCfgCfgGfuGfgUfuGf 1269 fgGfdTsdTuGfgdTsdT 169 169-187 caCfaAfcCfaCfcGfcGfcAfcG 1270GfCfcGfuGfcGfcGfgUfgGfuUf 1271 fgCfdTsdT gUfgdTsdT 170 170-188acAfaCfcAfcCfgCfgCfaCfgG 1272 GfGfcCfgUfgCfgCfgGfuGfgUf 1273 fcCfdTsdTuGfudTsdT 247 247-265 auGfcGfaCfcCfuCfcGfgGfa 1274CfCfgUfcCfcGfgAfgGfgUfcGf 1275 CfgGfdTsdT cAfudTsdT 248 248-266ugCfgAfcCfcUfcCfgGfgAfc 1276 GfCfcGfuCfcCfgGfaGfgGfuCf 1277 GfgCfdTsdTgCfadTsdT 249 249-267 gcGfaCfcCfuCfcGfgGfaCfg 1278GfGfcCfgUfcCfcGfgAfgGfgUf 1279 GfcCfdTsdT cGfcdTsdT 251 251-269gaCfcCfuCfcGfgGfaCfgGfc 1280 CfCfgGfcCfgUfcCfcGfgAfgGf 1281 CfgGfdTsdTgUfcdTsdT 252 252-270 acCfcUfcCfgGfgAfcGfgCfc 1282CfCfcGfgCfcGfuCfcCfgGfaGf 1283 GfgGfdTsdT gGfudTsdT 254 254-272ccUfcCfgGfgAfcGfgCfcGfg 1284 GfCfcCfcGfgCfcGfuCfcCfgGf 1285 GfgCfdTsdTaGfgdTsdT 329 329-347 agAfaAfgUfuUfgCfcAfaGfg 1286GfUfgCfcUfuGfgCfaAfaCfuUf 1287 CfaCfdTsdT uCfudTsdT 330 330-348gaAfaGfuUfuGfcCfaAfgGfc 1288 CfGfuGfcCfuUfgGfcAfaAfcUf 1289 AfcGfdTsdTuUfcdTsdT 332 332-350 aaGfuUfuGfcCfaAfgGfcAfc 1290CfUfcGfuGfcCfuUfgGfcAfaAf 1291 GfaGfdTsdT cUfudTsdT 333 333-351agUfuUfgCfcAfaGfgCfaCfg 1292 AfCfuCfgUfgCfcUfuGfgCfaAf 1293 AfgUfdTsdTaCfudTsdT 334 334-352 guUfuGfcCfaAfgGfcAfcGfa 1294UfAfcUfcGfuGfcCfuUfgGfcAf 1295 GfuAfdTsdT aAfcdTsdT 335 335-353uuUfgCfcAfaGfgCfaCfgAfg 1296 UfUfaCfuCfgUfgCfcUfuGfgCf 1297 UfaAfdTsdTaAfadTsdT 336 336-354 uuGfcCfaAfgGfcAfcGfaGfu 1298GfUfuAfcUfcGfuGfcCfuUfgGf 1299 AfaCfdTsdT cAfadTsdT 337 337-355ugCfcAfaGfgCfaCfgAfgUfa 1300 UfGfuUfaCfuCfgUfgCfcUfuGf 1301 AfcAfdTsdTgCfadTsdT 338 338-356 gcCfaAfgGfcAfcGfaGfuAfa 1302UfUfgUfuAfcUfcGfuGfcCfuUf 1303 CfaAfdTsdT gGfcdTsdT 361 361-379acGfcAfgUfuGfgGfcAfcUfu 1304 CfAfaAfaGfuGfcCfcAfaCfuGf 1305 UfuGfdTsdTcGfudTsdT 362 362-380 cgCfaGfuUfgGfgCfaCfuUfu 1306UfCfaAfaAfgUfgCfcCfaAfcUf 1307 UfgAfdTsdT gCfgdTsdT 363 363-381gcAfgUfuGfgGfcAfcUfuUfu 1308 UfUfcAfaAfaGfuGfcCfcAfaCf 1309 GfaAfdTsdTuGfcdTsdT 364 364-382 caGfuUfgGfgCfaCfuUfuUfg 1310CfUfuCfaAfaAfgUfgCfcCfaAf 1311 AfaGfdTsdT cUfgdTsdT 365 365-383agUfuGfgGfcAfcUfuUfuGfa 1312 UfCfuUfcAfaAfaGfuGfcCfcAf 1313 AfgAfdTsdTaCfudTsdT 366 366-384 guUfgGfgCfaCfuUfuUfgAfa 1314AfUfcUfuCfaAfaAfgUfgCfcCf 1315 GfaUfdTsdT aAfcdTsdT 367 367-385uuGfgGfcAfcUfuUfuGfaAfg 1316 GfAfuCfuUfcAfaAfaGfuGfcCf 1317 AfuCfdTsdTcAfadTsdT 368 368-386 ugGfgCfaCfuUfuUfgAfaGfa 1318UfGfaUfcUfuCfaAfaAfgUfgCf 1319 UfcAfdTsdT cCfadTsdT 369 369-387ggGfcAfcUfuUfuGfaAfgAfu 1320 AfUfgAfuCfuUfcAfaAfaGfuGf 1321 CfaUfdTsdTcCfcdTsdT 377 377-395 uuGfaAfgAfuCfaUfuUfuCfu 1322CfUfgAfgAfaAfaUfgAfuCfuUf 1323 CfaGfdTsdT cAfadTsdT 379 379-397gaAfgAfuCfaUfuUfuCfuCfa 1324 GfGfcUfgAfgAfaAfaUfgAfuCf 1325 GfcCfdTsdTuUfcdTsdT 380 380-398 aaGfaUfcAfuUfuUfcUfcAfg 1326AfGfgCfuGfaGfaAfaAfuGfaUf 1327 CfcUfdTsdT cUfudTsdT 385 385-403caUfuUfuCfuCfaGfcCfuCfc 1328 UfCfuGfgAfgGfcUfgAfgAfaA 1329 AfgAfdTsdTfaUfgdTsdT 394 394-412 agCfcUfcCfaGfaGfgAfuGfu 1330UfGfaAfcAfuCfcUfcUfgGfaGf 1331 UfcAfdTsdT gCfudTsdT 396 396-414ccUfcCfaGfaGfgAfuGfuUfc 1332 AfUfuGfaAfcAfuCfcUfcUfgGf 1333 AfaUfdTsdTaGfgdTsdT 397 397-415 cuCfcAfgAfgGfaUfgUfuCfa 1334UfAfuUfgAfaCfaUfcCfuCfuGf 1335 AfuAfdTsdT gAfgdTsdT 401 401-419agAfgGfaUfgUfuCfaAfuAfa 1336 CfAfgUfuAfuUfgAfaCfaUfcCf 1337 CfuGfdTsdTuCfudTsdT 403 403-421 agGfaUfgUfuCfaAfuAfaCfu 1338CfAfcAfgUfuAfuUfgAfaCfaUf 1339 GfuGfdTsdT cCfudTsdT 407 407-425ugUfuCfaAfuAfaCfuGfuGfa 1340 AfCfcUfcAfcAfgUfuAfuUfgAf 1341 GfgUfdTsdTaCfadTsdT 409 409-427 uuCfaAfuAfaCfuGfuGfaGfg 1342CfCfaCfcUfcAfcAfgUfuAfuUf 1343 UfgGfdTsdT gAfadTsdT 410 410-428ucAfaUfaAfcUfgUfgAfgGfu 1344 AfCfcAfcCfuCfaCfaGfuUfaUf 1345 GfgUfdTsdTuGfadTsdT 411 411-429 caAfuAfaCfuGfuGfaGfgUfg 1346GfAfcCfaCfcUfcAfcAfgUfuAf 1347 GfuCfdTsdT uUfgdTsdT 412 412-430aaUfaAfcUfgUfgAfgGfuGfg 1348 GfGfaCfcAfcCfuCfaCfaGfuUf 1349 UfcCfdTsdTaUfudTsdT 413 413-431 auAfaCfuGfuGfaGfgUfgGfu 1350AfGfgAfcCfaCfcUfcAfcAfgUf 1351 CfcUfdTsdT uAfudTsdT 414 414-432uaAfcUfgUfgAfgGfuGfgUfc 1352 AfAfgGfaCfcAfcCfuCfaCfaGf 1353 CfuUfdTsdTuUfadTsdT 416 416-434 acUfgUfgAfgGfuGfgUfcCfu 1354CfCfaAfgGfaCfcAfcCfuCfaCf 1355 UfgGfdTsdT aGfudTsdT 418 418-436ugUfgAfgGfuGfgUfcCfuUfg 1356 UfCfcCfaAfgGfaCfcAfcCfuCf 1357 GfgAfdTsdTaCfadTsdT 419 419-437 guGfaGfgUfgGfuCfcUfuGfg 1358UfUfcCfcAfaGfgAfcCfaCfcUf 1359 GfaAfdTsdT cAfcdTsdT 425 425-443ugGfuCfcUfuGfgGfaAfuUfu 1360 UfCfcAfaAfuUfcCfcAfaGfgAf 1361 GfgAfdTsdTcCfadTsdT 431 431-449 uuGfgGfaAfuUfuGfgAfaAfu 1362GfUfaAfuUfuCfcAfaAfuUfcCf 1363 UfaCfdTsdT cAfadTsdT 432 432-450ugGfgAfaUfuUfgGfaAfaUfu 1364 GfGfuAfaUfuUfcCfaAfaUfuCf 1365 AfcCfdTsdTcCfadTsdT 433 433-451 ggGfaAfuUfuGfgAfaAfuUfa 1366AfGfgUfaAfuUfuCfcAfaAfuUf 1367 CfcUfdTsdT cCfcdTsdT 434 434-452ggAfaUfuUfgGfaAfaUfuAfc 1368 UfAfgGfuAfaUfuUfcCfaAfaUf 1369 CfuAfdTsdTuCfcdTsdT 458 458-476 agAfgGfaAfuUfaUfgAfuCfu 1370GfAfaAfgAfuCfaUfaAfuUfcCf 1371 UfuCfdTsdT uCfudTsdT 459 459-477gaGfgAfaUfuAfuGfaUfcUfu 1372 GfGfaAfaGfaUfcAfuAfaUfuCf 1373 UfcCfdTsdTcUfcdTsdT 463 463-481 aaUfuAfuGfaUfcUfuUfcCfu 1374AfGfaAfgGfaAfaGfaUfcAfuAf 1375 UfcUfdTsdT aUfudTsdT 464 464-482auUfaUfgAfuCfuUfuCfcUfu 1376 AfAfgAfaGfgAfaAfgAfuCfaUf 1377 CfuUfdTsdTaAfudTsdT 466 466-484 uaUfgAfuCfuUfuCfcUfuCfu 1378UfUfaAfgAfaGfgAfaAfgAfuCf 1379 UfaAfdTsdT aUfadTsdT 468 468-486ugAfuCfuUfuCfcUfuCfuUfa 1380 CfUfuUfaAfgAfaGfgAfaAfgAf 1381 AfaGfdTsdTuCfadTsdT 471 471-489 ucUfuUfcCfuUfcUfuAfaAfg 1382GfGfuCfuUfuAfaGfaAfgGfaAf 1383 AfcCfdTsdT aGfadTsdT 476 476-494ccUfuCfuUfaAfaGfaCfcAfu 1384 UfGfgAfuGfgUfcUfuUfaAfgA 1385 CfcAfdTsdTfaGfgdTsdT 477 477-495 cuUfcUfuAfaAfgAfcCfaUfc 1386CfUfgGfaUfgGfuCfuUfuAfaGf 1387 CfaGfdTsdT aAfgdTsdT 479 479-497ucUfuAfaAfgAfcCfaUfcCfa 1388 UfCfcUfgGfaUfgGfuCfuUfuAf 1389 GfgAfdTsdTaGfadTsdT 481 481-499 uuAfaAfgAfcCfaUfcCfaGfg 1390CfCfuCfcUfgGfaUfgGfuCfuUf 1391 AfgGfdTsdT uAfadTsdT 482 482-500uaAfaGfaCfcAfuCfcAfgGfa 1392 AfCfcUfcCfuGfgAfuGfgUfcUf 1393 GfgUfdTsdTuUfadTsdT 492 492-510 ccAfgGfaGfgUfgGfcUfgGfu 1394AfUfaAfcCfaGfcCfaCfcUfcCf 1395 UfaUfdTsdT uGfgdTsdT 493 493-511caGfgAfgGfuGfgCfuGfgUfu 1396 CfAfuAfaCfcAfgCfcAfcCfuCf 1397 AfuGfdTsdTcUfgdTsdT 494 494-512 agGfaGfgUfgGfcUfgGfuUfa 1398AfCfaUfaAfcCfaGfcCfaCfcUfc 1399 UfgUfdTsdT CfudTsdT 495 495-513ggAfgGfuGfgCfuGfgUfuAfu 1400 GfAfcAfuAfaCfcAfgCfcAfcCf 1401 GfuCfdTsdTuCfcdTsdT 496 496-514 gaGfgUfgGfcUfgGfuUfaUfg 1402GfGfaCfaUfaAfcCfaGfcCfaCfc 1403 UfcCfdTsdT UfcdTsdT 497 497-515agGfuGfgCfuGfgUfuAfuGfu 1404 AfGfgAfcAfuAfaCfcAfgCfcAf 1405 CfcUfdTsdTcCfudTsdT 499 499-517 guGfgCfuGfgUfuAfuGfuCfc 1406UfGfaGfgAfcAfuAfaCfcAfgCf 1407 UfcAfdTsdT cAfcdTsdT 520 520-538gcCfcUfcAfaCfaCfaGfuGfg 1408 GfCfuCfcAfcUfgUfgUfuGfaGf 1409 AfgCfdTsdTgGfcdTsdT 542 542-560 uuCfcUfuUfgGfaAfaAfcCfu 1410UfGfcAfgGfuUfuUfcCfaAfaGf 1411 GfcAfdTsdT gAfadTsdT 543 543-561ucCfuUfuGfgAfaAfaCfcUfg 1412 CfUfgCfaGfgUfuUfuCfcAfaAf 1413 CfaGfdTsdTgGfadTsdT 550 550-568 gaAfaAfcCfuGfcAfgAfuCfa 1414UfGfaUfgAfuCfuGfcAfgGfuU 1415 UfcAfdTsdT fuUfcdTsdT 551 551-569aaAfaCfcUfgCfaGfaUfcAfu 1416 CfUfgAfuGfaUfcUfgCfaGfgUf 1417 CfaGfdTsdTuUfudTsdT 553 553-571 aaCfcUfgCfaGfaUfcAfuCfa 1418CfUfcUfgAfuGfaUfcUfgCfaGf 1419 GfaGfdTsdT gUfudTsdT 556 556-574cuGfcAfgAfuCfaUfcAfgAfg 1420 UfUfcCfuCfuGfaUfgAfuCfuGf 1421 GfaAfdTsdTcAfgdTsdT 586 586-604 gaAfaAfuUfcCfuAfuGfcCfu 1422CfUfaAfgGfcAfuAfgGfaAfuUf 1423 UfaGfdTsdT uUfcdTsdT 587 587-605aaAfaUfuCfcUfaUfgCfcUfu 1424 GfCfuAfaGfgCfaUfaGfgAfaUf 1425 AfgCfdTsdTuUfudTsdT 589 589-607 aaUfuCfcUfaUfgCfcUfuAfg 1426CfUfgCfuAfaGfgCfaUfaGfgAf 1427 CfaGfdTsdT aUfudTsdT 592 592-610ucCfuAfuGfcCfuUfaGfcAfg 1428 AfGfaCfuGfcUfaAfgGfcAfuAf 1429 UfcUfdTsdTgGfadTsdT 593 593-611 ccUfaUfgCfcUfuAfgCfaGfu 1430AfAfgAfcUfgCfuAfaGfgCfaUf 1431 CfuUfdTsdT aGfgdTsdT 594 594-612cuAfuGfcCfuUfaGfcAfgUfc 1432 UfAfaGfaCfuGfcUfaAfgGfcAf 1433 UfuAfdTsdTuAfgdTsdT 596 596-614 auGfcCfuUfaGfcAfgUfcUfu 1434GfAfuAfaGfaCfuGfcUfaAfgGf 1435 AfuCfdTsdT cAfudTsdT 597 597-615ugCfcUfuAfgCfaGfuCfuUfa 1436 AfGfaUfaAfgAfcUfgCfuAfaGf 1437 UfcUfdTsdTgCfadTsdT 598 598-616 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UfGfaUfgGfaGfgUfgCfaGfuUf 1885 fcAfdTsdTuUfudTsdT 1328 1328-1346 acUfgCfaCfcUfcCfaUfcAfg 1886CfCfaCfuGfaUfgGfaGfgUfgCf 1887 UfgGfdTsdT aGfudTsdT 1332 1332-1350caCfcUfcCfaUfcAfgUfgGfc 1888 AfUfcGfcCfaCfuGfaUfgGfaGf 1889 GfaUfdTsdTgUfgdTsdT 1333 1333-1351 acCfuCfcAfuCfaGfuGfgCfg 1890GfAfuCfgCfcAfcUfgAfuGfgAf 1891 AfuCfdTsdT gGfudTsdT 1335 1335-1353cuCfcAfuCfaGfuGfgCfgAfu 1892 GfAfgAfuCfgCfcAfcUfgAfuGf 1893 CfuCfdTsdTgAfgdTsdT 1338 1338-1356 caUfcAfgUfgGfcGfaUfcUfc 1894GfUfgGfaGfaUfcGfcCfaCfuGf 1895 CfaCfdTsdT aUfgdTsdT 1344 1344-1362ugGfcGfaUfcUfcCfaCfaUfc 1896 CfAfgGfaUfgUfgGfaGfaUfcGf 1897 CfuGfdTsdTcCfadTsdT 1345 1345-1363 ggCfgAfuCfuCfcAfcAfuCfc 1898GfCfaGfgAfuGfuGfgAfgAfuC 1899 UfgCfdTsdT fgCfcdTsdT 1346 1346-1364gcGfaUfcUfcCfaCfaUfcCfu 1900 GfGfcAfgGfaUfgUfgGfaGfaUf 1901 GfcCfdTsdTcGfcdTsdT 1347 1347-1365 cgAfuCfuCfcAfcAfuCfcUfg 1902CfGfgCfaGfgAfuGfuGfgAfgA 1903 CfcGfdTsdT fuCfgdTsdT 1348 1348-1366gaUfcUfcCfaCfaUfcCfuGfcC 1904 CfCfgGfcAfgGfaUfgUfgGfaGf 1905 fgGfdTsdTaUfcdTsdT 1353 1353-1371 ccAfcAfuCfcUfgCfcGfgUfg 1906UfGfcCfaCfcGfgCfaGfgAfuGf 1907 GfcAfdTsdT uGfgdTsdT 1354 1354-1372caCfaUfcCfuGfcCfgGfuGfg 1908 AfUfgCfcAfcCfgGfcAfgGfaUf 1909 CfaUfdTsdTgUfgdTsdT 1355 1355-1373 acAfuCfcUfgCfcGfgUfgGfc 1910AfAfuGfcCfaCfcGfgCfaGfgAf 1911 AfuUfdTsdT uGfudTsdT 1357 1357-1375auCfcUfgCfcGfgUfgGfcAfu 1912 UfAfaAfuGfcCfaCfcGfgCfaGf 1913 UfuAfdTsdTgAfudTsdT 1360 1360-1378 cuGfcCfgGfuGfgCfaUfuUfa 1914CfCfcUfaAfaUfgCfcAfcCfgGf 1915 GfgGfdTsdT cAfgdTsdT 1361 1361-1379ugCfcGfgUfgGfcAfuUfuAfg 1916 CfCfcCfuAfaAfuGfcCfaCfcGf 1917 GfgGfdTsdTgCfadTsdT 1362 1362-1380 gcCfgGfuGfgCfaUfuUfaGfg 1918AfCfcCfcUfaAfaUfgCfcAfcCf 1919 GfgUfdTsdT gGfcdTsdT 1363 1363-1381ccGfgUfgGfcAfuUfuAfgGfg 1920 CfAfcCfcCfuAfaAfuGfcCfaCf 1921 GfuGfdTsdTcGfgdTsdT 1366 1366-1384 guGfgCfaUfuUfaGfgGfgUfg 1922AfGfuCfaCfcCfcUfaAfaUfgCf 1923 AfcUfdTsdT cAfcdTsdT 1369 1369-1387gcAfuUfuAfgGfgGfuGfaCfu 1924 AfGfgAfgUfcAfcCfcCfuAfaAf 1925 CfcUfdTsdTuGfcdTsdT 1370 1370-1388 caUfuUfaGfgGfgUfgAfcUfc 1926AfAfgGfaGfuCfaCfcCfcUfaAf 1927 CfuUfdTsdT aUfgdTsdT 1371 1371-1389auUfuAfgGfgGfuGfaCfuCfc 1928 GfAfaGfgAfgUfcAfcCfcCfuAf 1929 UfuCfdTsdTaAfudTsdT 1372 1372-1390 uuUfaGfgGfgUfgAfcUfcCfu 1930UfGfaAfgGfaGfuCfaCfcCfcUf 1931 UfcAfdTsdT aAfadTsdT 1373 1373-1391uuAfgGfgGfuGfaCfuCfcUfu 1932 GfUfgAfaGfgAfgUfcAfcCfcCf 1933 CfaCfdTsdTuAfadTsdT 1374 1374-1392 uaGfgGfgUfgAfcUfcCfuUfc 1934UfGfuGfaAfgGfaGfuCfaCfcCf 1935 AfcAfdTsdT cUfadTsdT 1404 1404-1422ucUfgGfaUfcCfaCfaGfgAfa 1936 CfAfgUfuCfcUfgUfgGfaUfcCf 1937 CfuGfdTsdTaGfadTsdT 1408 1408-1426 gaUfcCfaCfaGfgAfaCfuGfg 1938UfAfuCfcAfgUfuCfcUfgUfgGf 1939 AfuAfdTsdT aUfcdTsdT 1409 1409-1427auCfcAfcAfgGfaAfcUfgGfa 1940 AfUfaUfcCfaGfuUfcCfuGfuGf 1941 UfaUfdTsdTgAfudTsdT 1411 1411-1429 ccAfcAfgGfaAfcUfgGfaUfa 1942GfAfaUfaUfcCfaGfuUfcCfuGf 1943 UfuCfdTsdT uGfgdTsdT 1412 1412-1430caCfaGfgAfaCfuGfgAfuAfu 1944 AfGfaAfuAfuCfcAfgUfuCfcUf 1945 UfcUfdTsdTgUfgdTsdT 1419 1419-1437 acUfgGfaUfaUfuCfuGfaAfa 1946GfGfuUfuUfcAfgAfaUfaUfcCf 1947 AfcCfdTsdT aGfudTsdT 1426 1426-1444auUfcUfgAfaAfaCfcGfuAfa 1948 CfCfuUfuAfcGfgUfuUfuCfaGf 1949 AfgGfdTsdTaAfudTsdT 1427 1427-1445 uuCfuGfaAfaAfcCfgUfaAfa 1950UfCfcUfuUfaCfgGfuUfuUfcAf 1951 GfgAfdTsdT gAfadTsdT 1430 1430-1448ugAfaAfaCfcGfuAfaAfgGfa 1952 AfUfuUfcCfuUfuAfcGfgUfuU 1953 AfaUfdTsdTfuCfadTsdT 1431 1431-1449 gaAfaAfcCfgUfaAfaGfgAfa 1954GfAfuUfuCfcUfuUfaCfgGfuUf 1955 AfuCfdTsdT uUfcdTsdT siRNA Sequence withChemical Modification Info lower case (n) = 2′-O-Me; Nf = 2′-F; dT= deoxy-T residue; s = phosphorothioate backbone modification; iB= inverted abasic

TABLE 7 AR Target Sequences SEQ ID ID Code Target Sequence NO:NM_000044.3 Exon Species XD- 17 CAAAGGUUCUCUGCUAGAC 1956 1987-2005 1 h01817K1 GACA XD- 27 UCUGGGUGUCACUAUGGAG 1957 2819-2837 2 h 01827K1 CUCUXD- 28 CUGGGUGUCACUAUGGAGC 1958 2820-2838 2 h 01828K1 UCUC XD- 29GGGUGUCACUAUGGAGCUC 1959 2822-2840 2 h 01829K1 UCAC XD-01821K1 21UACUACAACUUUCCACUGGC 1960 2207-2225 1 h UCU XD-01825K1 25AAGCUUCUGGGUGUCACUA 1961 2814-2832 2 h, m UGGA XD-01826K1 26CUUCUGGGUGUCACUAUGG 1962 2817-2835 2 h AGCU

TABLE 8 β-catenin Target Sequences Generic R # name Gene Targetsequences R- 1797mfm CTNNB1 CUGUUGGAUUGAU SEQ ID UUUCGAAUCAAUCCA SEQ ID1146 UCGAAAUU NO: ACAGUU NO: 1963 1964 R- 1870mfm CTNNB1 ACGACUAGUUCAGUSEQ ID AAGCAACUGAACUAG SEQ ID 1147 UGCUUUU NO: UCGUUU NO: 1965 1966

TABLE 9 β-catenin and β-catenin associated siRNA Sequences Sense StrandSequence SEQ Antisense Strand SEQ Generic (5′-3′) ID Sequence (5′-3′) IDR # name Gene Passenger Strand (PS)2 NO: Guide Strand (GS)3 NO: R-1797mfm CTNNB1 iBcsuGfuUfgGfaUfuGfa 1967 usUfsusCfgAfaUfcAfaUfcC 19681146 UfuCfgAfaAfusuiB faAfcAfgusu R- 1870mfm CTNNB1 iBascGfaCfuAfgUfuCfa1969 asAfsgsCfaAfcUfgAfaCfuA 1970 1147 GfuUfgCfuUfusuiB fgUfcGfuusu R-PA1746 1746 GCUCAAAGCAAUUU 1971 UGUAGAAAUUGCUUU 1972 1150 CUACAdTsdTGAGCdTsdT R- PA2328 2328 GGAUGAAACACAAA 1973 UACCUUUUGUGUUUCA 1974 1151AGGUAdTsdT UCCdTsdT R- PA2522 2522 UGUCAGAGUUACUG 1975 UGAAACAGUAACUCUG1976 1152 UUUCAdTsdT ACAdTsdT R- PA3484 3484 AGCAAGAACAGAAA 1977UUUUAUUUCUGUUCU 1978 1153 UAAAAdTsdT UGCUdTsdT R- PA5018 5018CUAGUUCAUUUCAA 1979 UAAUUUUGAAAUGAA 1980 1154 AAUUAdTsdT CUAGdTsdT R-PB183 183 CAAGUUCACAAUUA 1981 UUGGGUAAUUGUGAA 1982 1155 CCCAAdTsdTCUUGdTsdT R- PB272 272 GCUUGAAGAUGAAA 1983 UCGUGUUUCAUCUUCA 1984 1156CACGAdTsdT AGCdTsdT R- PB862 862 AGAUCAAGAAAAUG 1985 UCAUACAUUUUCUUGA1986 1157 UAUGAdTsdT UCUdTsdT R- PB948 948 CCAAAGAAAACACG 1987UAAUUCGUGUUUUCU 1988 1158 AAUUAdTsdT UUGGdTsdT R- PB1520 1520CUUCGAUAAGAUUA 1989 UUCAAUAAUCUUAUCG 1990 1159 UUGAAdTsdT AAGdTsdT R-Myc953U 953 AGGAACUAUGACCU 1991 AGUCGAGGUCAUAGU 1992 1160 CGACUdTsdTUCCUdTsdT R- Myc622U 622 ACGACGAGACCUUC 1993 UUGAUGAAGGUCUCG 1994 1161AUCAAdTsdT UCGUdTsdT R- Myc1370U 1370 AAGAUGAGGAAGAA 1995UCGAUUUCUUCCUCAU 1996 1162 AUCGAdTsdT CUUdTsdT R- Myc1364U 1364AGGAAGAAAUCGAU 1997 ACAACAUCGAUUUCUU 1998 1163 GUUGUdTsdT CCUdTsdT R-Myc1711U 1711 AGCUUUUUUGCCCU 1999 CACGCAGGGCAAAAAA 2000 1164 GCGUGdTsdTGCUdTsdT R- Myc1769U 1769 AGGUAGUUAUCCUU 2001 UUUUUAAGGAUAACU 2002 1165AAAAAdTsdT ACCUdTsdT siRNA Sequence with Chemical Modification Infolower case (n) = 2′-O-Me; Nf = 2′-F; dT = deoxy-T residue; s= phosphorothioate backbone modification; iB = inverted abasic

TABLE 10 PIK3CA* and PIK3CB* Target Sequences Gene Gene SEQ ID Symbol IDName Target Sequences (97-mer) NO: PIK3CA 5290 PIK3CA_1746TGCTGTTGACAGTGAGCGCCAGCTCAAAGCAATTT 2003CTACATAGTGAAGCCACAGATGTATGTAGAAATTG CTTTGAGCTGTTGCCTACTGCCTCGGA PIK3CA5290 PIK3CA_2328 TGCTGTTGACAGTGAGCGAAAGGATGAAACACAAA 2004AGGTATAGTGAAGCCACAGATGTATACCTTTTGTGT TTCATCCTTCTGCCTACTGCCTCGGA PIK3CA5290 PIK3CA_2522 TGCTGTTGACAGTGAGCGCCATGTCAGAGTTACTG 2005TTTCATAGTGAAGCCACAGATGTATGAAACAGTAA CTCTGACATGATGCCTACTGCCTCGGA PIK3CA5290 PIK3CA_3555 TGCTGTTGACAGTGAGCGCAACTAGTTCATTTCAA 2006AATTATAGTGAAGCCACAGATGTATAATTTTGAAA TGAACTAGTTTTGCCTACTGCCTCGGA PIK3CA5290 PIK3CA_3484 TGCTGTTGACAGTGAGCGCACAGCAAGAACAGAAA 2007TAAAATAGTGAAGCCACAGATGTATTTTATTTCTGT TCTTGCTGTATGCCTACTGCCTCGGA PIK3CB5291 PIK3CB_862 TGCTGTTGACAGTGAGCGACAAGATCAAGAAAATG 2008TATGATAGTGAAGCCACAGATGTATCATACATTTTC TTGATCTTGCTGCCTACTGCCTCGGA PIK3CB5291 PIK3CB_183 TGCTGTTGACAGTGAGCGCAGCAAGTTCACAATTA 2009CCCAATAGTGAAGCCACAGATGTATTGGGTAATTG TGAACTTGCTTTGCCTACTGCCTCGGA PIK3CB5291 PIK3CB_1520 TGCTGTTGACAGTGAGCGCCCCTTCGATAAGATTAT 2010TGAATAGTGAAGCCACAGATGTATTCAATAATCTT ATCGAAGGGATGCCTACTGCCTCGGA PIK3CB5291 PIK3CB_272 TGCTGTTGACAGTGAGCGAGAGCTTGAAGATGAAA 2011CACGATAGTGAAGCCACAGATGTATCGTGTTTCAT CTTCAAGCTCCTGCCTACTGCCTCGGA PIK3CB5291 PIK3CB_948 TGCTGTTGACAGTGAGCGACACCAAAGAAAACACG 2012AATTATAGTGAAGCCACAGATGTATAATTCGTGTTT TCTTTGGTGGTGCCTACTGCCTCGGA *Speciesis Homo sapiens.

TABLE 11 PIK3CA and PIK3CB siRNA Sequences SEQ SEQ Gene Gene ID IDSymbol ID Name siRNA Guide NO: siRNA passenger NO: PIK3CA 5290PIK3CA_1746 UGUAGAAAUUGCUU 2013 AGCUCAAAGCAAUUU 2014 UGAGCUGU CUACAUAPIK3CA 5290 PIK3CA_2328 UACCUUUUGUGUUU 2015 AGGAUGAAACACAAA 2016CAUCCUUC AGGUAUA PIK3CA 5290 PIK3CA_2522 UGAAACAGUAACUC 2017AUGUCAGAGUUACUG 2018 UGACAUGA UUUCAUA PIK3CA 5290 PIK3CA_3555UAAUUUUGAAAUGA 2019 ACUAGUUCAUUUCAA 2020 ACUAGUUU AAUUAUA PIK3CA 5290PIK3CA_3484 UUUUAUUUCUGUUC 2021 CAGCAAGAACAGAAA 2022 UUGCUGUA UAAAAUAPIK3CB 5291 PIK3CB_862 UCAUACAUUUUCUU 2023 AAGAUCAAGAAAAUG 2024 GAUCUUGCUAUGAUA PIK3CB 5291 PIK3CB_183 UUGGGUAAUUGUGA 2025 GCAAGUUCACAAUUA 2026ACUUGCUU CCCAAUA PIK3CB 5291 PIK3CB_1520 UUCAAUAAUCUUAU 2027CCUUCGAUAAGAUUA 2028 CGAAGGGA UUGAAUA PIK3CB 5291 PIK3CB_272UCGUGUUUCAUCUU 2029 AGCUUGAAGAUGAAA 2030 CAAGCUCC CACGAUA PIK3CB 5291PIK3CB_948 UAAUUCGUGUUUUC 2031 ACCAAAGAAAACACG 2032 UUUGGUGG AAUUAUA

TABLE 12 Additional polynucleic acid molecule sequences Base SEQ startID SEQ position Guide strand NO: Passenger strand ID NO: EGFR 333ACUCGUGCCUUGGCAA 2082 AGUUUGCCAAGGCACGAG 2083 R1246 ACUUU UUU EGFR 333ACUCGUGCCUUGGCAA 2084 AGUUUGCCAAGGCACGAG 2085 R1195 ACUUU UUU EGFR 333ACUCGUGCCUUGGCAA 2086 AGUUUGCCAAGGCACGAG 2087 R1449 ACUUU UUU KRAS 237UGAAUUAGCUGUAUCG 2088 TGACGAUACAGCUAAUUC 2089 R1450 UCAUU AUU KRAS 237UGAAUUAGCUGUAUCG 2090 UGACGAUACAGCUAAUUC 2091 R1443 UCAUU AUU KRAS 237UGAAUUAGCUGUAUCG 2092 UGACGAUACAGCUAAUUC 2093 R1194 UCAUU AUU CTNNB11248 UAAGUAUAGGUCCUCA 2094 UAAUGAGGACCUAUACU 2095 R1442 UUAUU UAUUCTNNB1 1797 TUUCGAAUCAAUCCAA 2096 CUGUUGGAUUGAUUCGA 2097 R1404 CAGUUAAUU CTNNB1 1797 UUUCGAAUCAAUCCAA 2098 CUGUUGGAUUGAUUCGA 2099 R1441CAGUU AAUU CTNNB1 1797 UUUCGAAUCAAUCCAA 2100 CUGUUGGAUUGAUUCGA 2101R1523 CAGUU AAUU HPRT 425 AUAAAAUCUACAGUCA 2102 CUAUGACUGUAGAUUUU 2103R1492 UAGUU AUUU HPRT 425 UUAAAAUCUACAGUCA 2104 CUAUGACUGUAGAUUUU 2105R1526 UAGUU AAUU HPRT 425 UUAAAAUCUACAGUCA 2106 CUAUGACUGUAGAUUUU 2107R1527 UAGUU AAUU AR 2822 GAGAGCUCCAUAGUGA 2108 GUGUCACUAUGGAGCUCU 2109R1245 CACUU CUU

Example 2. General Experimental Protocol

Stem-Loop qPCR Assay for Quantification of siRNA

Plasma samples were directly diluted in TE buffer. 50 mg tissue pieceswere homogenized in 1 mL of Trizol using a FastPrep-24 tissuehomogenizer (MP Biomedicals) and then diluted in TE buffer. Standardcurves were generated by spiking siRNA into plasma or homogenized tissuefrom untreated animals and then serially diluting with TE buffer. Theantisense strand of the siRNA was reverse transcribed using a TaqManMicroRNA reverse transcription kit (Applied Biosystems) with 25 nM of asequence-specific stem-loop RT primer. The cDNA from the RT step wasutilized for real-time PCR using TaqMan Fast Advanced Master Mix(Applied Biosystems) with 1.5 μM of forward primer, 0.75 μM of reverseprimer, and 0.2 μM of probe. The sequences of KRAS and EGFR siRNAantisense strands and all primers and probes used to measure them areshown in Table 13. Quantitative PCR reactions were performed usingstandard cycling conditions in a ViiA 7 Real-Time PCR System (LifeTechnologies). The Ct values were transformed into plasma or tissueconcentrations using the linear equations derived from the standardcurves.

TABLE 13 Sequences for all siRNA antisense strands, primers, and probesused in the stem-loop qPCR assay. SEQ Target Name Sequence (5′-3′) IDNO: KRAS Antisense UGAAUUAGCUGUAUCGUCAUU 2033 KRAS RTGTCGTATCCAGTGCAGGGTCCGAGG 2034 TATTCGCACTGGATACGACAATGACG KRAS ForwardGGCGGCTGAATTAGCTGTATCGT 2035 KRAS Reverse AGTGCAGGGTCCGAG 2036 KRASProbe (6FAM)-TGGATACGACAATGAC- 2037 (NFQ-MGB) EGFR AntisenseACUCGUGCCUUGGCAAACUUU 2038 EGFR RT GTCGTATCCAGTGCAGGGTCCGAGGT 2039ATTCGCACTGGATACGACAAAGTTTG EGFR Forward GGCGGCACTCGTGCCTTGGCA 2040 EGFRReverse AGTGCAGGGTCCGAG 2041 EGFR Probe (6FAM)-TGGATACGACAAAGTT- 2042(NFQ-MGB)

Comparative qPCR Assay for Determination of mRNA Knockdown

Tissue samples were homogenized in Trizol as described above. Total RNAwas isolated using RNeasy RNA isolation 96-well plates (Qiagen), then500 ng RNA was reverse transcribed with a High Capacity RNA to cDNA kit(ThermoFisher). KRAS, EGFR, CTNNB1 and PPIB mRNA was quantified byTaqMan qPCR analysis performed with a ViiA 7 Real-Time PCR System. TheTaqMan primers and probes for KRAS were designed and validated byAvidity and are shown in Table 14. The TaqMan primers and probes forEGFR and CTNNB1 were purchased from Applied Biosystems as pre-validatedgene expression assays. PPIB (housekeeping gene) was used as an internalRNA loading control, with all TaqMan primers and probes for PPIBpurchased from Applied Biosystems as pre-validated gene expressionassays. Results are calculated by the comparative Ct method, where thedifference between the target gene (KRAS, CTNNB1, or EGFR) Ct value andthe PPIB Ct value (ΔCt) is calculated and then further normalizedrelative to the PBS control group by taking a second difference (i\ Ct).

TABLE 14 Sequences of primers and probes for KRAS mRNA detection usingcomparative qPCR assay. SEQ ID Target Species Name Sequence (5′-3′) NO:KRAS Mouse Forward CGCCTTGACGATACAGCTAAT 2043 KRAS Mouse ReverseTGTTTCCTGTAGGAGTCCTCT 2044 AT KRAS Mouse Probe (6FAM)- 2045TCACTTTGT(Zen)GGATGAGT and ATGACCCTACG-(IABkFQ) 2114 KRAS Human ForwardGTGCCTTGACGATACAGCTAAT 2046 KRAS Human Reverse CCAAGAGACAGGTTTCTCCATC2047 KRAS Human Probe (6FAM)- 2048 CCAACAATA(Zen)GAGGATTC andCTACAGGAAGCA-(IABkFQ) 2115

Animals

All animal studies were conducted following protocols in accordance withthe Institutional Animal Care and Use Committee (IACUC) at ExploraBioLabs, which adhere to the regulations outlined in the USDA AnimalWelfare Act as well as the “Guide for the Care and Use of LaboratoryAnimals” (National Research Council publication, 8th Ed., revised in2011). All mice were obtained from either Charles River Laboratories orHarlan Laboratories.

H358, HCC827, and Hep-3B2 1-7 Subcutaneous Flank Tumor Model

For the H358 subcutaneous flank tumor model, tumor cells were inoculatedand tumors were established according to the following methods. FemaleNCr nu/nu mice were identified by ear-tag the day before cell injection.Mice were weighed prior to inoculation. H358 cells were cultured with10% FBS/RAMI medium and harvested with 0.05% Trypsin and Cell Stripper(MediaTech). 5 million H358 cells in 0.05 ml Hank's Balanced SaltSolution (HBSS) with Matrigel (1:1) were injected subcutaneously (SC)into the upper right flank of each mouse. Tumor growth was monitored bytumor volume measurement using a digital caliper starting on day 7 afterinoculation, and followed 2 times per week until average tumor volumereaches >100 & ≤300 mm³. Once tumors were staged to the desired volume(average from 100 to 300 mm³), animals were randomized and mice withvery large or small tumors were culled. Mice were divided into therequired groups and randomized by tumor volume. Mice were then treatedas described in the individual experiments.

For the Hep3B orthotropic liver tumor model, tumor cells were inoculatedand tumors were established according to the following methods. FemaleNCr nu/nu mice were identified by ear-tag the day before, mice will beanesthetized with isoflurane. The mice were then placed in a supineposition on a water circulating heating pad to maintain bodytemperature. A small transverse incision below the sternum will be madeto expose the liver. Cancer cells were slowly injected into the upperleft lobe of the liver using a 28-gauge needle. The cells were injectedat a 30-degree angle into the liver, so that a transparent bleb of cellscan be seen through the liver capsule. Hep 3B2.1 7 cells were preparedby suspending in cold PBS (0.1-5×10⁶ cells) and mixing with dilutedmatrigel (30× in PBS). 30-50 ul of the cell/matrigel was inoculated.After injection, a small piece of sterile gauze was placed on theinjection site, and light pressure was applied for 1 min to preventbleeding. The abdomen was then closed with a 6-0 silk suture. Aftertumor cell implantation, animals were kept in a warm cage, observed for1-2 h, and subsequently returned to the animal room after full recoveryfrom the anesthesia. 7-10 days after tumor implantation animals wererandomized, divided into the required groups and then treated asdescribed in the individual experiments.

LNCap Subcutaneous Flank Tumor Model

LNCaP cells (ATCC® CRL-1740™) were grown in RPMI+10% FBS supplementedwith non-essential amino acids and sodium pyruvate to a confluency ofabout 80%. Cells were mixed 1:1 with matrigel and 5-7*106 cells injectedsubcutaneously into male SCID mice (6-8 weeks). Tumors usually developedwithin 3-5 weeks to a size of 100-350 mm³. Animals bearing tumors withinthis range were randomized and treated with ASCs by injections into thetail vein. For PD studies animals were sacrificed 96 hours afterinjection and organ fragments harvested, weighted, and frozen in liquidnitrogen. For RNA isolation, organ samples were homogenized in Trizoland RNA prepared using a Qiagen RNeasy 96 Plus kit following theinstructions by the manufacturer. RNA concentrations were determinedspectroscopically. RNAs were converted into cDNAs by reversetranscription and expression of specific targets quantified by qPCRusing the ΔΔCT method and validated Taqman assays (Thermofisher).Samples were standardize to the expression levels of PPIB.

Cholesterol siRNA Conjugate Synthesis

All siRNA single strands were fully assembled on solid phase usingstandard phospharamidite chemistry and purified over HPLC. Purifiedsingle strands were duplexed to get the double stranded siRNA. Structureof cholesterol conjugated to the passenger strand is illustrated in FIG.2. Table 15 shows KRAS, EGFR, and CTNNB1 siRNA sequences.

TABLE 15 SEQ MW ID siRNA Strand Sequence (5′-3′) observed NO: KRASPassenger Chol-iBusgAfcGfaUfaC 7813.6 2049 faGfcUfaAfuUfcAfusui B KRASGuide UfsGfsasAfuUfaGfcUfg 6874.6 2050 UfaUfcGfuCfausu EGFR PassengerChol-iBasgUfuUfgCfcA 7884.6 2051 faGfgCfaCfgAfgUfusuiB EGFR GuideasCfsusCfgUfgCfcUfuG 6860.6 2052 fgCfaAfaCfuusu CTNNB1 PassengerChol-iBcsuGfuUfgGfaU 7847.5 2053 fuGfaUfuCfgAfaAfusui B CTNNB1 GuideusUfsusCfgAfaUfcAfaU 6852.6 2054 fcCfaAfcAfgusu

The siRNA chemical modifications include:

-   -   upper case (N)=2′-OH (ribo);    -   lower case (n)=2′-O-Me (methyl);    -   dN=2′-H (deoxy);    -   Nf=2′-F (fluoro);    -   s=phosphorothioate backbone modification;    -   iB=inverted abasic

Peptide Synthesis

Peptides were synthesized on solid phase using standard Fmoc chemistry.Both peptides have cysteine at the N-terminus and the cleaved peptideswere purified by HPLC and confirmed by mass spectroscopy. INF7 peptideis as illustrated in FIG. 3 (SEQ ID NO: 2055). Melittin peptide is asillustrated in FIG. 4 (SEQ ID NO: 2060).

Anti-EGFR Antibody

Anti-EGFR antibody is a fully human IgG1κ monoclonal antibody directedagainst the human epidermal growth factor receptor (EGFR). It isproduced in the Chinese Hamster Ovary cell line DJT33, which has beenderived from the CHO cell line CHO-K1SV by transfection with a GS vectorcarrying the antibody genes derived from a human anti-EGFR antibodyproducing hybridoma cell line (2F8). Standard mammalian cell culture andpurification technologies are employed in the manufacturing of anti-EGFRantibody.

The theoretical molecular weight (MW) of anti-EGFR antibody withoutglycans is 146.6 kDa. The experimental MW of the major glycosylatedisoform of the antibody is 149 kDa as determined by mass spectrometry.Using SDS-PAGE under reducing conditions the MW of the light chain wasfound to be approximately 25 kDa and the MW of the heavy chain to beapproximately 50 kDa. The heavy chains are connected to each other bytwo inter-chain disulfide bonds, and one light chain is attached to eachheavy chain by a single inter-chain disulfide bond. The light chain hastwo intra-chain disulfide bonds and the heavy chain has four intra-chaindisulfide bonds. The antibody is N-linked glycosylated at Asn305 of theheavy chain with glycans composed of N-acetyl-glucosamine, mannose,fucose and galactose. The predominant glycans present are fucosylatedbi-antennary structures containing zero or one terminal galactoseresidue.

The charged isoform pattern of the IgG1κ antibody has been investigatedusing imaged capillary IEF, agarose IEF and analytical cation exchangeHPLC. Multiple charged isoforms are found, with the main isoform havingan isoelectric point of approximately 8.7.

The major mechanism of action of anti-EGFR antibody is a concentrationdependent inhibition of EGF-induced EGFR phosphorylation in A431 cancercells. Additionally, induction of antibody-dependent cell-mediatedcytotoxicity (ADCC) at low antibody concentrations has been observed inpre-clinical cellular in vitro studies.

Example 3: Synthesis, Purification and Analysis of Antibody-PEG-EGFR andAntibody-EGFR Conjugates—Conjugation Scheme 1

Step 1: Antibody Conjugation with Maleimide-PEG-NHS Followed by SH-EGFR

Anti-EGFR antibody (EGFR-Ab) was exchanged with 1× Phosphate buffer (pH7.4) and made up to 5 mg/ml concentration. To this solution, 2equivalents of SMCC linker or maleimide-PEGxkDa-NHS (x=1, 5, 10, 20) wasadded and rotated for 4 hours at room temperature. Unreactedmaleimide-PEG was removed by spin filtration using 50 kDa MWCO Amiconspin filters and PBS pH 7.4. The antibody-PEG-Mal conjugate wascollected and transferred into a reaction vessel. SH-C6-EGFR (2equivalents) was added at RT to the antibody-PEG-maleimide in PBS androtated overnight, see FIG. 88. The reaction mixture was analyzed byanalytical SAX column chromatography and conjugate along with unreactedantibody and siRNA was seen.

Step 2: Purification

The crude reaction mixture was purified by AKTA explorer FPLC usinganion exchange chromatography method-1. Fractions containing theantibody-PEG-EGFR conjugate were pooled, concentrated and bufferexchanged with PBS, pH 7.4. Antibody siRNA conjugates with SMCC linker,PEG1 kDa, PEG5 kDa and PEG10 kDa were separated based on the siRNAloading. Conjugates with PEG20 kDa gave poor separation.

Step-3: Analysis of the Purified Conjugate

The isolated conjugate was characterized by either mass spec orSDS-PAGE. The purity of the conjugate was assessed by analytical HPLCusing either anion exchange chromatography method-2 or anion exchangechromatography method-3. Examples of all the conjugates made using thesemethods are described in Table 16.

TABLE 16 List of AXCYB conjugates HPLC retention time (minutes) withAnion exchange chromatography method-2 Conjugate DAR = 1 DAR = 2 DAR= >2 EGFR-Ab-EGFR 9.0 9.9 10.4 EGFR-Ab-PEG1kDa-EGFR 9.2 10.0 10.6EGFR-Ab-PEG5kDa-EGFR 8.7 9.3 ND EGFR-Ab-PEG10kDa-EGFR 8.6 8.8 to 10; mixof DAR 2-3 EGFR-Ab-PEG20kDa-EGFR 8.6; Mixture of DAR of 1-3 Holo-anti-Bcell Ab- 9.2  9.5 PEG20kDa-EGFR

Anion Exchange Chromatography Method-1

-   1. Column: Tosoh Bioscience, TSKGel SuperQ-5PW, 21.5 mm ID×15 cm, 13    um-   2. Solvent A: 20 mM TRIS buffer, pH 8.0; Solvent B: 20 mM TRIS, 1.5    M NaCl, pH 8.0; Flow Rate: 6.0 ml/min-   3. Gradient:

a. % A % B Column Volume b. 100 0 1.00 c. 60 40 18.00 d. 40 60 2.00 e.40 60 5.00 f. 0 100 2.00 g. 100 0 2.00

Anion Exchange Chromatography Method-2

-   1. Column: Thermo Scientific, ProPac™ SAX-10, Bio LC™, 4×250 mm-   2. Solvent A: 80% 10 mM TRIS pH 8, 20% ethanol; Solvent B: 80% 10 mM    TRIS pH 8, 20% ethanol, 1.5 M NaCl; Flow Rate: 1.0 ml/min-   3. Gradient:

a. Time % A % B b. 0.0 90 10 c. 3.00 90 10 d. 11.00 40 60 e. 13.00 40 60f. 15.00 90 10 g. 20.00 90 10

Anion Exchange Chromatography Method-3

-   1. Column: Thermo Scientific, ProPac™ SAX-10, Bio LC™, 4×250 mm-   2. Solvent A: 80% 10 mM TRIS pH 8, 20% ethanol; Solvent B: 80% 10 mM    TRIS pH 8, 20% ethanol, 1.5 M NaCl-   3. Flow Rate: 0.75 ml/min-   4. Gradient:

a. Time % A % B b. 0.0 90 10 c. 3.00 90 10 d. 11.00 40 60 e. 23.00 40 60f. 25.00 90 10 g. 30.00 90 10

The analytical data for EGFR antibody-PEG20 kDa-EGFR are illustrated inFIG. 5 and FIG. 6. FIG. 5 shows the analytical HPLC of EGFRantibody-PEG20 kDa-EGFR. FIG. 6 shows a SDS-PAGE analysis of EGFRantibody-PEG20 kDa-EGFR conjugate. The analytical chromatogram of EGFRantibody-PEG10 kDa-EGFR is illustrated in FIG. 7. The analytical datafor EGFR antibody-PEG5 kDa-EGFR are illustrated in FIG. 8 and FIG. 9.FIG. 8 shows the analytical chromatogram of EGFR antibody-PEG5 kDa-EGFR.FIG. 9 shows SDS PAGE analysis of EGFR antibody-PEG10 kDa-EGFR and EGFRantibody-PEG5 kDa-EGFR conjugates. The analytical data for EGFRantibody-PEG1 kDa-EGFR conjugates with different siRNA loading isillustrated in FIG. 10.

Example 4: Synthesis, Purification and Analysis of Antibody-siRNA-PEGConjugates—Conjugation Scheme-2

Step 1: Antibody Conjugation with SMCC Linker Followed by SH-KRAS-PEG5kDa

Anti-EGFR antibody was exchanged with 1× Phosphate buffer (pH 7.4) andmade up to 5 mg/ml concentration. To this solution, 2 equivalents ofSMCC linker (succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate) was added and rotatedfor 4 hours at room temperature, see FIG. 89. Unreacted SMCC linker wasremoved by spin filtration using 50 kDa MWCO Amicon spin filters and PBSbuffer pH 7.4. The retentate was collected and 2 equivalents ofSH-C6-KRAS-PEG5 kDa was added at RT and rotated overnight. The reactionmixture was analyzed by analytical SAX column chromatography and theconjugate along with unreacted antibody and siRNA was observed.

Step 2: Purification

The crude reaction mixture was purified by AKTA explorer FPLC usinganion exchange chromatography method-1. Fractions containing theantibody-KRAS-PEG conjugate were pooled, concentrated and bufferexchanged with PBS, pH 7.4.

Step-3: Analysis of the Purified Conjugate

The isolated conjugate was characterized by either mass spec orSDS-PAGE. The purity of the conjugate was assessed by analytical HPLCusing anion exchange chromatography method-3 (described in example 1).Examples of the conjugates made using the methods described in Examples4 and 5 are illustrated in Table 17.

TABLE 17 List of A-X-B-Y-C conjugates HPLC retention time (minutes) withAnion exchange chromatography method-3 Conjugate DAR = 1 DAR = 2 DAR= >2 EGFR-Ab-KRAS-PEG5kDa 9.2 EGFR-Ab-S-S-KRAS- 9.0 PEG5kDa Holo-anti-Bcell 9.2 9.7 10.1 Ab-KRAS-PEG5kDa Panitumumab-KRAS- 9.2 9.7 10.2 PEG5kDa

The HPLC chromatogram of EGFR Antibody-KRAS-PEG5 kDa is illustrated inFIG. 11. The HPLC chromatogram of Panitumumab-KRAS-PEG5 kDa is as shownin FIG. 12.

Example 5: Synthesis, Purification and Analysis ofAntibody-S—S-siRNA-PEG Conjugates—Conjugation Scheme-3

Step 1: Antibody Conjugation with SPDP Linker Followed by SH-siRNA-PEG5kDa

Anti-EGFR antibody was exchanged with 1× Phosphate buffer (pH 7.4) andmade up to 5 mg/ml concentration. To this solution, 2 equivalents ofSPDP linker (succinimidyl 3-(2-pyridyldithio)propionate) was added androtated for 4 hours at room temperature. Unreacted SPDP linker wasremoved by spin filtration using 50 kDa MWCO Amicon spin filters and pH7.4 PBS buffer. The retentate was collected and 2 equivalents ofSH-C6-siRNA-PEG5 kDa was added at room temperature and rotatedovernight, see FIG. 90. The reaction mixture was analyzed by analyticalSAX column chromatography and conjugate along with unreacted antibodyand siRNA was seen.

Step 2: Purification

The crude reaction mixture was purified by AKTA explorer FPLC usinganion exchange chromatography method-1. Fractions containing theantibody-PEG-siRNA conjugate were pooled, concentrated and bufferexchanged with PBS, pH 7.4.

Step-3: Analysis of the Purified Conjugate

The isolated conjugate was characterized by either mass spec orSDS-PAGE. The purity of the conjugate was assessed by analytical HPLCusing anion exchange chromatography method-2. The HPLC chromatogram ofEGFR Antibody-S—S-siRNA-PEG5 kDa (DAR=1) is as shown in FIG. 13.

Example 6: Synthesis, Purification and Analysis ofAntibody-SMCC-Endosomal Escape Peptide Conjugates—Conjugation Scheme-4

Step 1: Antibody Conjugation with SMCC Linker or Maleimide-PEG-NHSFollowed by SH-Cys-Peptide-CONH₂

Anti-EGFR antibody was exchanged with 1× Phosphate buffer (pH 7.4) andmade up to 10 mg/ml concentration. To this solution, 3 equivalents ofSMCC linker (succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate) or maleimide-PEG 1kDa-NHS was added and rotated for 1.5 hours at room temperature.Unreacted SMCC linker or PEG linker was removed by spin filtration using50 kDa MWCO Amicon spin filters and PBS buffer pH 7.4 (25 mM MES pH=6.1for Melittin conjugates). The retentate was collected and 3 equivalentsof SH-Cys-Peptide-CONH₂ was added at RT and rotated overnight. See FIG.91. The reaction mixture was then purified by either HIC chromatographyor cation exchange chromatography to isolate the anti-EGFRantibody-Peptide or anti-EGFR antibody-PEG1k-Peptide.

Step 2: Purification

The crude reaction mixture was purified by AKTA explorer FPLC usingeither hydrophobic interaction chromatography (HIC) method-1 or cationexchange chromatography method-1. Fractions containing theantibody-peptide conjugates were pooled, concentrated and bufferexchanged with PBS, pH 7.4 (10 mM Acetate pH=6.0 for Melittinconjugates).

Step-3: Analysis of the Purified Conjugate

The isolated conjugate was characterized by either mass spec orSDS-PAGE. Purity and peptide loading was assessed by analytical HPLCusing either HIC method-2 or cation exchange chromatography method-2.Examples of all the conjugates made using the method of Example 6 aredescribed in Tables 18 and 19.

TABLE 18 List of AXYD conjugates HPLC retention time (minutes) with HICmethod-2 Conjugate DAR = 1 DAR = 2 DAR = >2 EGFR-Ab-INF7 7.7 9.3 11.2EGFR-Ab-PEG24-INF7 8.4 12.2 15.2

TABLE 19 List of AXYD conjugates HPLC retention time (minutes) withcation exchange chromatography method-2 Conjugate DAR = 1 DAR = >1 DAR= >2 EGFR-Ab-Melittin 40.9 54.8 EGFR-Ab-PEG1kDa- 48. 53.4 55.8 melittin

Cation Exchange Chromatography Method-1

-   1. Column: GE Healthcare HiPrep SP HP 16/10-   2. Solvent A: 50 mM MES pH=6.0; Solvent B: 50 mM MES+0.5M NaCl    pH=6.0; Flow Rate: 2.0 ml/min-   3. Gradient:

a. % A % B Column Volume b. 100 0 0.1 c. 100 0 Flush loop 12 ml d. 100 02.5 e. 0 100 15 f. 0 100 5 g. 100 0 0.5 h. 100 0 5

Cation Exchange Chromatography Method-2

-   1. Column: Thermo Scientific, MAbPac™ SCX-10, Bio LC^(Tm), 4×250 mm    (product #074625)-   2. Solvent A: 20 mM MES pH=5.5; Solvent B: 20 mM MES+0.3 M NaCl    pH=5.5; Flow Rate: 0.5 ml/min-   3. Gradient:

a. Time % A % B b. 0.0 100 0 c. 5 100 0 d. 50 0 100 e. 80 0 100 f. 85100 0 g. 90 100 0

Hydrophobic Interaction Chromatography Method-1 (HIC Method-1)

-   1. Column: GE Healthcare Butyl Sepharose High Performance    (17-5432-02) 200 ml-   2. Solvent A: 50 mM Sodium Phosphate+0.8M ammonium sulfate (pH=7.0);    Solvent B: 80% 50 mM Sodium Phosphate (pH=7.0), 20% IPA; Flow Rate:    3.0 ml/min-   3. Gradient:

a. % A % B Column Volume b. 100 0 0.1 c. 0 100 3 d. 0 100 1.35 e. 100 00.1 f. 100 0 0.5

Hydrophobic Interaction Chromatography Method-2 (HIC Method-2)

-   1. Column: Tosoh Bioscience TSKgel Butyl-NPR 4.6 mm ID×10 cm 2.5 μm-   2. Solvent A: 100 mM Sodium phosphate+1.8 M ammonium sulfate    (pH=7.0); Solvent B: 80% 100 mM sodium phosphate (pH=7.0), 20% IPA;    Flow Rate: 0.5 ml/min-   3. Gradient:

a. Time % A % B b. 0 100 0 c. 3 50 50 d. 21 0 100 e. 23 0 100 f. 25 1000

FIG. 14 illustrates the HPLC chromatogram of EGFRantibody-PEG24-Melittin (loading=˜1). FIG. 15 illustrates the HPLCchromatogram of EGFR antibody-Melittin (n=˜0.1). FIG. 16 shows the massspectrum of EGFR antibody-Melittin (n=1). FIG. 17 shows the HICchromatogram of EGFR antibody-PEG1 kDa-INF7 (Peptide loading=˜1). FIG.18 shows the HPLC chromatogram of EGFR antibody-INF7 (PeptideLoading=˜1).

Example 7: Synthesis, Purification and Analysis ofEEP-Antibody-siRNA-PEG Conjugates—Conjugation Scheme-5

Step 1: Conjugation of PEG24 Linker Followed by SH-Cys-Peptide-CONH₂ toEGFR-Ab-siRNA-PEG

EGFR-Ab-siRNA-PEG conjugate with a siRNA loading of 1 was conjugatedwith 4 equivalents of PEG1k linker (succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate) in PBS, pH 7.4 bufferand rotated for 1.5 hours at room temperature. See FIG. 92. UnreactedPEG1k linker was removed by spin filtration using 50 kDa MWCO Amiconspin filters and PBS buffer pH 7.4. The retentate was collected and 4equivalents of SH-Cys-Peptide-CONH₂ was added at RT and rotatedovernight.

Step 2: Purification

The reaction mixture was then purified by repeated spin filtration usingPBS buffer pH7.4 and 50 kDa Amicon spin filters until the unreactedpeptide was removed as monitored by HPLC. The product contains a mixtureof conjugates with 0, 1, 2, 3 or more peptides conjugated to theantibody backbone.

Step-3: Analysis of the Purified Conjugate

The isolated conjugate was characterized by either mass spec orSDS-PAGE. The purity and the peptide loading of the conjugate wasassessed by analytical HPLC using either HIC method-2 or cation exchangechromatography method-2. Examples of the conjugates made using themethod described in Example 7 are shown in Table 20.

TABLE 20 List of (A-X-B-Y-Cn)-L-D conjugates HPLC retention time(minutes) with cation exchange chromatography method-2 DAR = DAR = DAR =DAR = Conjugate 0 1 2 3 (EGFR-Ab-siRNA-PEG5kDa)- 24 38 27 9 PEG1k-INF7(EGFR-Ab-siRNA-PEG5kDa)- 24 11.79 (broad peak) PEG1k-melittin

FIG. 19 shows INF7-PEG1 kDa-(EGFR antibody-KRAS-PEG5 kDa). FIG. 20 showsMelittin-PEG1 kDa-(EGFR antibody-KRAS-PEG5 kDa).

Example 8: In Vivo Pharmacokinetics Study of a EGFR Antibody-siRNA-PEGConjugate (PK-055)

Groups (n=3) of female NCr nu/nu mice bearing subcutaneous flank H358tumors 100-150 mm³ in volume were treated with one intravenous (i.v.)tail vein injection of siRNA conjugate, while control groups (n=4) ofthe same mice received one i.v. injection of PBS as a vehicle control.Treatment groups that received EGFR antibody-siRNA-PEG conjugates weredosed at 0.5 mg/kg (based on the weight of siRNA) and groups thatreceived cholesterol-siRNA conjugates were dosed at 15 mg/kg. All groups(treatments and controls) were administered a dose volume of 5 mL/kg.Non-terminal blood samples were collected at 2, 15, or 60 minutespost-dose via puncture of the retro-orbital plexus and centrifuged togenerate plasma for PK analysis. Mice were sacrificed by CO₂asphyxiation at 24, 96, or 168 h post-dose. Table 21 describes the studydesign in more detail and provides a cross-reference to the conjugatesynthesis and characterization. Terminal blood samples were collectedvia cardiac puncture and processed to generate plasma for PK analysis.50 mg pieces of tumor, liver, kidney, and lung were collected andsnap-frozen in liquid nitrogen. mRNA knockdown analysis and siRNAquantitation were performed as described in Examples 2-7.

TABLE 21 Study design for a EGFR antibody-siRNA-PEG Conjugate (PK-055)with a cross-reference to the synthesis and characterization of theconjugates tested. siRNA:EGFR- siRNA Ab melittin:siRNA Survival TerminalHarvest Cross-reference Test Dose Ratio Ratio Dose Bleed Bleed Time tosynthesis and Group Article N (mg/kg) (mol/mol) (mol/mol) ROA Schedule(min) (h) (h) characterization 4 EGFR- 3 0.5 1.4 — IV t = 0 2 24 24Example 3 5 Ab- 3 0.5 1.4 — IV t = 0 15 96 96 6 PEG10k- 3 0.5 1.4 — IV t= 0 60 168 168 EGFR 7 EGFR- 3 0.5 1.25 — IV t = 0 2 24 24 Example 3 8Ab- 3 0.5 1.25 — IV t = 0 15 96 96 9 PEG5k- 3 0.5 1.25 — IV t = 0 60 168168 EGFR 10 EGFR- 3 0.5 1.25 — IV t = 0 2 24 24 Example 3 11 Ab- 3 0.51.25 — IV t = 0 15 96 96 12 PEG1k- 3 0.5 1.25 — IV t = 0 60 168 168 EGFR13 EGFR- 3 0.5 1.3 — IV t = 0 2 24 24 Example 3 14 Ab- 3 0.5 1.3 — IV t= 0 15 96 96 15 EGFR 3 0.5 1.3 — IV t = 0 60 168 168 16 EGFR- 3 0.5 2.6— IV t = 0 2 24 24 Example 4 17 Ab- 3 0.5 2.6 — IV t = 0 15 96 96 18KRAS- 3 0.5 2.6 — IV t = 0 60 168 168 PEG5k (n = 2 siRNAs per EGFR- Ab)19 EGFR- 3 0.5 1.0 — IV t = 0 2 24 24 Example 4 20 Ab- 3 0.5 1.0 — IV t= 0 15 96 96 21 KRAS- 3 0.5 1.0 — IV t = 0 60 168 168 PEG5k (n = 1 siRNAper EGFR- Ab) 22 EGFR- 3 0.5 1.0 1:1 IV t = 0 2 24 24 Example 4 and 6 23Ab- 3 0.5 1.0 1:1 IV t = 0 15 96 96 24 KRAS- 3 0.5 1.0 1:1 IV t = 0 60168 168 PEG5k (n = 1) + EGFR- Ab- melittin 25 Chol- 3 15 — — IV t = 0 224 24 General 26 EGFR- 3 15 — — IV t = 0 15 96 96 experimental 27 333mfm3 15 — — IV t = 0 60 168 168 (Example 2) 28 Chol- 3 15 — — IV t = 0 2 2424 General 29 KRAS- 3 15 — — IV t = 0 15 96 96 experimental 30 237ffm 315 — — IV t = 0 60 168 168 (Example 2) 31 Vehicle 4 — — — IV t = 0 — —24 32 4 — — — IV t = 0 — — 96 33 4 — — — IV t = 0 — — 168

PEG linkers of various molecular weights and a small molecule linkerwere used to attach EGFR siRNA to an EGFR antibody (EGFR-Ab) and the PKwas assessed to determine the effect of the linker molecular weight onthe behavior of the mAb-siRNA conjugate in plasma. As illustrated inFIG. 21, the molecular weight of the PEG linker does not have a largeimpact on the plasma PK, except for the 10 kDa PEG leads to a fastersiRNA clearance (i.e. lower plasma concentrations at later times). Theorientation of the siRNA and PEG relative to the EGFR-Ab was alsoexplored. As illustrated in FIG. 22, having the siRNA in between theEGFR-Ab and the PEG5k (EGFR antibody-KRAS-PEG5k) results insignificantly higher plasma concentrations than the alternativeconjugate where PEG5k is in between the EGFR-Ab and the siRNA (EGFRantibody-PEG5k-EGFR). In some instances, the use of two different siRNAson these conjugates does not impact the plasma kinetics.

The drug loading on the EGFR-Ab was also investigated, with n=1 and n=2siRNAs per EGFR-Ab. As illustrated in FIG. 23, having only one siRNA perEGFR-Ab resulted in much higher plasma concentrations, whereas thehigher loading of n=2 siRNA per EGFR-Ab resulted in faster clearancefrom plasma. The impact of adding an endosomal escape peptide (melittin)was assessed. EGFR antibody-KRAS-PEG5k and EGFR antibody-melittin weremixed together in solution and co-injected. As illustrated in FIG. 24,the presence of EGFR antibody-melittin increases the clearance fromplasma of EGFR antibody-KRAS-PEG5k at later times.

The plasma PK of cholesterol-siRNA conjugates was next compared to themAb-siRNA conjugates after intravenous administration via tail veininjection. As illustrated in FIG. 25, the chol-siRNA conjugates arecleared much faster from plasma than the mAb-siRNA conjugates. Asillustrated from the PK profile, having either EGFR or KRAS siRNA on theconjugate did not affect the plasma kinetics.

In addition to the plasma PK analysis, siRNA concentrations weredetermined in tissues at various times post-dose to determine the tissuePK. Tissue concentrations were measured pmol/g and then converted topmol/mL by assuming the density of tissue equals 1 g/mL. In FIG. 26, aconcentration of 1 nM=1 nmol/L=1 pmol/mL=1 pmol/g tissue. As illustratedin FIG. 26A, a single i.v. dose of 0.5 mg/kg of EGFR antibody-siRNAresulted in approximately 100 nM concentrations of siRNA in tumor at 24h post-dose for virtually all of the conjugates. In the case of theseEGFR antibody-linker-siRNA conjugates, the molecular weight of thelinker between the EGFR-Ab and the EGFR siRNA does not seem to alter thePK of these conjugates in the s.c. flank H358 tumors. As illustrated inFIG. 26B, the concentration of siRNA in liver following a single i.v.dose of 0.5 mg/kg of EGFR antibody-siRNA is approximately 100 nM at 24 hpost-dose, similar to that seen in tumor. Only the small molecule linkerat 24 h post-dose produces a siRNA concentration in liver approximatelyhalf of what is seen with longer PEG linkers. siRNA concentrationsdecrease over time in both tumor and liver tissue with these EGFRantibody-linker-siRNA conjugates.

The orientation of the siRNA and PEG relative to the EGFR-Ab was alsoexplored relative to the tissue PK profiles. As illustrated in FIG. 27,both the EGFR antibody-KRAS-PEG5k and the EGFR antibody-PEG5k-EGFRconjugates deliver approximately 100 nM siRNA into both tumor and liverfollowing a single i.v. dose of 0.5 mg/kg. However, while the EGFRantibody-KRAS-PEG5k maintains the siRNA concentration in tumor atapproximately 100 nM until 168 h post-dose, the other 3 curves declinein concentration over time. Next, the tissue PK as a function of drugloading was assessed. As illustrated from FIG. 28, n=1 siRNA per EGFR-Abdelivered higher amounts of siRNA into tumor compared to liver. However,increasing the siRNA loading to n=2 siRNA per EGFR-Ab increased theamount of siRNA delivered to liver and decreased the amount of siRNAdelivered to tumor. Additionally, EGFR antibody-melittin was mixed withsome formulations in order to introduce endosomal escape functionality.As illustrated from FIG. 29, mixing and co-administering EGFRantibody-melittin with EGFR antibody-siRNA did not have a large impacton the tissue PK. The addition of melittin decreased uptake of siRNA intumor and increased the uptake of siRNA in liver.

The tissue PK profiles of cholesterol-siRNA conjugates (using both EGFRand KRAS siRNA) in liver and in s.c. flank H358 tumors was alsoassessed. As illustrated from FIG. 30, both chol-siRNA conjugatesdelivered approximately 5 μM concentrations of siRNA into liver 24 hfollowing a single i.v. dose of 15 mg/kg. In liver, the chol-KRASappears to clear slightly faster than the chol-EGFR on the 1-week timescale. The two different chol-siRNA conjugates further show different PKprofiles in tumor. Both cholesterol conjugates deliver less siRNA intotumor compared to liver, but the chol-EGFR delivers more siRNA intotumor when compared to the chol-KRAS conjugate. Both chol-siRNAconjugates are cleared from tumor over time and with a similar slope.

A PD analysis followed the PK analysis. As illustrated in FIG. 31A, thechol-KRAS conjugate produced only marginal (˜25%) mRNA knockdown of theKRAS target gene in tumor following a single i.v. dose of 15 mg/kg.However, as illustrated in FIG. 31B, the same 15 mg/kg dose of chol-KRASwas able to produce >50% mRNA knockdown in the mouse liver. Thechol-EGFR conjugate was able to produce >50% mRNA knockdown in tumor, asillustrated in FIG. 32. In some instances, the higher knockdown withchol-EGFR in tumor compared to chol-KRAS is due to the higher siRNAconcentrations observed in tumor with chol-EGFR compared to chol-KRAS(FIG. 30). Finally, as illustrated in FIGS. 33 and 34, most of the EGFRantibody-siRNA conjugates resulted in approximately 25-50% EGFR or KRASmRNA knockdown in tumors after a single IV dose, but at a much lowerdose (0.5 mg/kg) compared to the chol-siRNA conjugates.

Example 9: Synthesis, Purification and Analysis of AdditionalAntibody-siRNA Conjugates—Scheme-6: Antibody-Lys-siRNA-PEG ConjugatesVia Antibody Lysine Conjugation of SMCC Linker

Step 1: Antibody Conjugation with SMCC Linker Followed by SH-siRNA

Antibody was buffer exchanged with 1× Phosphate buffer (pH 7.4) and madeup to 10 mg/ml concentration. To this solution, 2 equivalents of SMCClinker dissolved in DMSO was added and rotated for 4 hours at roomtemperature. Unreacted SMCC linker was removed by spin filtration using50 kDa MWCO Amicon spin filters and PBS pH 7.4. The antibody-maleimideconjugate was collected into a reaction vessel and SH-C6-siRNA orSH-C6-siRNA-C6-NHCO-PEG-XkDa (2 equivalents) (X=0.5 kDa to 10 kDa) wasadded at RT in pH 7.4 PBS with 5 mM EDTA and rotated overnight. See FIG.93. Analysis of the reaction mixture by analytical SAX columnchromatography method-2 showed antibody siRNA conjugate along withunreacted antibody and siRNA.

Step 2: Purification

The crude reaction mixture was purified by AKTA explorer FPLC usinganion exchange chromatography method-1. Fractions containing DAR1 andDAR>2 antibody-siRNA-PEG conjugates were separated, concentrated andbuffer exchanged with pH 7.4 PBS.

Step-3: Analysis of the Purified Conjugate

The isolated conjugates were characterized by SAX chromatography, SECchromatography and SDS-PAGE analysis. The purity of the conjugate wasassessed by analytical HPLC using either anion exchange chromatographymethod-2. All DAR1 conjugate generally eluted at 9.0±0.4 minutes whilethe DAR2 and DAR3 conjugates generally eluted at 9.7±0.2 minutes.Typical DAR1 conjugate is greater than 90% pure after purification whiletypical DAR>2 lysine conjugates contains 70-80% DAR2 and 20-30% DAR3.

Scheme-7: Antibody-Cys-siRNA-PEG Conjugates Via Antibody CysteineConjugation

Step 1: Antibody Interchain Disulfide Reduction with TCEP

Antibody was buffer exchanged with borax buffer (pH 8) and made up to 10mg/ml concentration. To this solution, 2 equivalents of TCEP in waterwas added and rotated for 2 hours at RT. The resultant reaction mixturewas buffer exchanged with pH 7.4 PBS containing 5 mM EDTA and added to asolution of SMCC-C6-siRNA or SMCC-C6-siRNA-C6-NHCO-PEG-XkDa (2equivalents) (X=0.5 kDa to 10 kDa) in pH 7.4 PBS containing 5 mM EDTA atRT and rotated overnight. Analysis of the reaction mixture by analyticalSAX column chromatography showed antibody siRNA conjugate along withunreacted antibody and siRNA. See FIG. 94

Step 2: Purification

The crude reaction mixture was purified by AKTA explorer FPLC usinganion exchange chromatography method-1. Fractions containing DAR1 andDAR>2 antibody-PEG-siRNA conjugates were separated, concentrated andbuffer exchanged with pH 7.4 PBS.

Step-3: Analysis of the Purified Conjugate

The isolated conjugates were characterized by SEC, SAX chromatographyand SDS-PAGE.

The purity of the conjugate was assessed by analytical HPLC using eitheranion exchange chromatography method-2 or anion exchange chromatographymethod-3. Isolated DAR1 conjugates are typically eluted at 9.0+0.3 minon analytical SAX method-2 and are greater than 90% pure. The typicalDAR>2 cysteine conjugate contains more than 85% DAR2 and less than 15%DAR3.

Scheme-8: Antibody siRNA Conjugates Via Antibody Inter-Chain CysteineConjugation

Step 1: Antibody Interchain Disulfide Reduction with TCEP

Antibody was buffer exchanged with borax buffer (pH 8) and made up to 10mg/ml concentration. To this solution, 2 equivalents of TCEP in waterwas added and rotated for 2 hours at RT. The resultant reaction mixturewas buffer exchanged with pH 7.4 PBS containing 5 mM EDTA and added to asolution of CBTF-C6-siRNA-C6-NHCO-PEG-5 kDa (2 equivalents) in pH 7.4PBS containing 5 mM EDTA at RT and rotated overnight. Analysis of thereaction mixture by analytical SAX column chromatography showed antibodysiRNA conjugate along with unreacted antibody and siRNA. See FIG. 95.

Step 2: Purification

The crude reaction mixture was purified by AKTA explorer FPLC usinganion exchange chromatography method-1. Fractions containing DAR1 andDAR>2 antibody-siRNA conjugates were separated, concentrated and bufferexchanged with pH 7.4 PBS. Typical DAR>2 cysteine conjugate containsgreater than 85% DAR2 and less than 15% DAR3 or higher.

Step-3: Analysis of the Purified Conjugate

The isolated conjugates were characterized by either mass spec orSDS-PAGE. The purity of the conjugate was assessed by analytical HPLCusing either anion exchange chromatography method-2 or anion exchangechromatography method-3.

Scheme-9: Antibody siRNA Conjugates Via Antibody Inter-Chain CysteineConjugation

Step 1: Antibody Reduction with TCEP

Antibody was buffer exchanged with borax buffer (pH 8) and made up to 5mg/ml concentration. To this solution, 2 equivalents of TCEP in waterwas added and rotated for 2 hours at RT. The resultant reaction mixturewas exchanged with pH 7.4 PBS containing 5 mM EDTA and added to asolution of MBS-C6-siRNA-C6-NHCO-PEG-5 kDa (2 equivalents) in pH 7.4 PBScontaining 5 mM EDTA at RT and rotated overnight. Analysis of thereaction mixture by analytical SAX column chromatography showed antibodysiRNA conjugate along with unreacted antibody and siRNA. See FIG. 96.

Step 2: Purification

The crude reaction mixture was purified by AKTA explorer FPLC usinganion exchange chromatography method-1. Fractions containing DAR1 andDAR>2 antibody-siRNA conjugates were separated, concentrated and bufferexchanged with pH 7.4 PBS. Typical DAR>2 cysteine conjugate containsgreater than 85% DAR2 and less than 15% DAR3 or higher.

Step-3: Analysis of the Purified Conjugate

The isolated conjugates were characterized by either mass spec orSDS-PAGE. The purity of the conjugate was assessed by analytical HPLCusing either anion exchange chromatography method-2 or anion exchangechromatography method-3.

Scheme-10: Antibody siRNA Conjugates Via Antibody Inter-Chain CysteineConjugation

Step 1: Antibody Reduction with TCEP

Antibody was buffer exchanged with borax buffer (pH 8) and made up to 5mg/ml concentration. To this solution, 2 equivalents of TCEP in waterwas added and rotated for 2 hours at RT. The resultant reaction mixturewas exchanged with pH 7.4 PBS containing 5 mM EDTA and added to asolution of MBS-C6-siRNA-C6-NHCO-PEG-5 kDa (2 equivalents) in pH 7.4 PBScontaining 5 mM EDTA at RT and rotated overnight. Analysis of thereaction mixture by analytical SAX column chromatography showed antibodysiRNA conjugate along with unreacted antibody and siRNA. See FIG. 97.

Step 2: Purification

The crude reaction mixture was purified by AKTA explorer FPLC usinganion exchange chromatography method-1. Fractions containing DAR1 andDAR>2 antibody-siRNA conjugates were separated, concentrated and bufferexchanged with pH 7.4 PBS. Typical DAR>2 cysteine conjugate containsgreater than 85% DAR2 and less than 15% DAR3 or higher.

Step-3: Analysis of the Purified Conjugate

The isolated conjugates were characterized by either mass spec orSDS-PAGE. The purity of the conjugate was assessed by analytical HPLCusing either anion exchange chromatography method-2 or anion exchangechromatography method-3.

Scheme-11: Synthesis of Antibody-Lysine-S—S-siRNA-PEG Conjugates

Step 1: Antibody Conjugation with SPDP Linker Followed by SH-siRNA-PEG5kDa

Antibody was buffer exchanged with pH 7.4 1×PBS and made up to 10 mg/mlconcentration. To this solution, 2 equivalents of SPDP linker[succinimidyl 3-(2-pyridyldithio)propionate] or its methylated versionwas added and rotated for 4 hours at room temperature. Unreacted SPDPlinker was removed by spin filtration using 50 kDa MWCO Amicon spinfilters and pH 7.4 PBS buffer. The retentate was collected and 2equivalents of SH-C6-siRNA-PEG5 kDa in pH 7.4 PBS was added at roomtemperature and rotated overnight. The reaction mixture was analyzed byanalytical SAX column chromatography and the conjugate along withunreacted antibody and siRNA was seen. See FIG. 98.

Step 2: Purification

The crude reaction mixture was purified by AKTA explorer FPLC usinganion exchange chromatography method-1. Fractions containing DAR1 andDAR>2 antibody-siRNA conjugates were separated, concentrated and bufferexchanged with pH 7.4 PBS. Typical DAR>2 lysine conjugate contains 70 to80% DAR2 and 20 to 30% DAR3 or higher.

Step-3: Analysis of the Purified Conjugate

The isolated conjugate was characterized by either mass spec orSDS-PAGE. The purity of the conjugate was assessed by analytical HPLCusing anion exchange chromatography method-2.

Scheme-12: Synthesis of Antibody-Cysteine-S—S-siRNA-PEG Conjugates

Step 1: Antibody Reduction and Conjugation with Pyridyldithio-siRNA-PEG5kDa

Antibody was buffer exchanged with pH 8.0 borax buffer and made up to 10mg/ml concentration. To this solution, 1.5 equivalents of TCEP was addedand the reaction mixture was rotated for 1 hour at room temperature.Unreacted TCEP was removed by spin filtration using 50 kDa MWCO Amiconspin filters and buffer exchanged with pH 7.4 PBS buffer. The retentatewas collected and 2 equivalents of pyridyldithio-C6-siRNA-PEG5 kDa in pH7.4 PBS was added at room temperature and rotated overnight. Thereaction mixture was analyzed by analytical SAX column chromatographyand conjugate along with unreacted antibody and siRNA was seen. See FIG.99.

Step 2: Purification

The crude reaction mixture was purified by AKTA explorer FPLC usinganion exchange chromatography method-1. Fractions containing DAR1 andDAR>2 antibody-siRNA conjugates were separated, concentrated and bufferexchanged with pH 7.4 PBS.

Step-3: Analysis of the Purified Conjugate

The isolated conjugate was characterized by either mass spec orSDS-PAGE. The purity of the conjugate was assessed by analytical HPLCusing anion exchange chromatography method-2. Typical DAR>2 cysteineconjugate contains 90% DAR2 and 10% DAR3 or higher.

Scheme-13: Synthesis of Antibody-Cysteine-ECL-siRNA-PEG Conjugates

Step 1: Antibody Reduction and Conjugation with Maleimide-ECL-siRNA-PEG5kDa

Antibody was buffer exchanged with pH 8.0 borax buffer and made up to 10mg/ml concentration. To this solution, 1.5 equivalents of TCEP(Tris(2-carboxyethyl)phosphine hydrochloride) reagent was added androtated for 1 hour at room temperature. Unreacted TCEP was removed byspin filtration using 50 kDa MWCO Amicon spin filters and pH 7.4 PBSbuffer with 5 mM EDTA. The retentate was collected and 1.5 equivalentsof maleimide-ECL-C6-siRNA-PEG5 kDa in pH 7.4 PBS was added at roomtemperature and rotated overnight. The reaction mixture was analyzed byanalytical SAX column chromatography and conjugate along with unreactedantibody and siRNA was seen. See FIG. 100.

Step 2: Purification

The crude reaction mixture was purified by AKTA explorer FPLC usinganion exchange chromatography method-1. Fractions containing DAR1 andDAR>2 antibody-siRNA conjugates were separated, concentrated and bufferexchanged with pH 7.4 PBS.

Step-3: Analysis of the Purified Conjugate

The isolated conjugate was characterized by either mass spec orSDS-PAGE. The purity of the conjugate was assessed by analytical HPLCusing anion exchange chromatography method-2. Typical DAR>2 lysineconjugate contains 70 to 80% DAR2 and 20 to 30% DAR3 or higher.

Scheme-14: Antibody Lysine Conjugation with TCO/Tetrazine Linker

Step 1: Antibody Conjugation with NHS-PEG4-TCO Followed byMethyltetrazine-PEG4-siRNA-Peg5 kDa

Antibody was buffer exchanged with pH 7.4 PBS and made up to 5 mg/mlconcentration. To this solution, 2 equivalents of NHS-PEG4-TCO linkerwas added and rotated for 4 hours at room temperature. Unreacted linkerwas removed by spin filtration using 50 kDa MWCO Amicon spin filters andpH 7.4 PBS. The retentate was collected and 2 equivalents ofmethyltetrazine-PEG4-siRNA-PEG5 kDa in pH 7.4 PBS was added at roomtemperature. The reaction mixture was analyzed by analytical SAX columnchromatography and the antibody-siRNA conjugate was seen along with theunreacted antibody and siRNA. See FIG. 101.

Step 2: Purification

The crude reaction mixture was purified by AKTA explorer FPLC usinganion exchange chromatography method-1. Fractions containing DAR1 andDAR>2 antibody-siRNA conjugates were separated, concentrated and bufferexchanged with pH 7.4 PBS. Typical DAR>2 lysine conjugate contains70-80% DAR2 and 20-30% DAR3 or higher.

Step-3: Analysis of the Purified Conjugate

The characterization and purity of the isolated conjugate wascharacterized by either mass spec or SDS-PAGE. The purity of theconjugate was assessed by analytical HPLC using anion exchangechromatography method-2.

Scheme-15: Site Specific Conjugation at Antibody Glycans

Step 1: Antibody Glycan Modification and Gal-N₃ Addition

Antibody was buffer exchanged with pH 6.0, 50 mM sodium phosphate bufferand treated with EndoS2 at 37° C. for 16 hrs. The reaction mixture wasbuffer exchanged into TBS buffer (20 mM Tris, 0.9% NaCl, pH 7.4) andUDP-GalNAz was added followed by MnCl₂, and Gal-T(Y289L) in 50 mM Tris,5 mM EDTA (pH 8). The final solution contained concentrations of 0.4mg/mL antibody, 10 mM MnCl₂, 1 mM UDP-GalNAz, and 0.2 mg/mL Gal-T(Y289L)and was incubated overnight at 30° C. See FIG. 102.

Step 2: DIBO-PEG-TCO Conjugation to Azide Modified Antibody

The reaction mixture from step-1 was buffer exchanged with PBS and 2equivalents of DIBO-PEG4-TCO linker was added and rotated for 6 hours atroom temperature. Unreacted linker was removed by spin filtration using50 kDa MWCO Amicon spin filters and pH 7.4 PBS. The retentate wascollected and used as is in step-3.

Step 3: Methyl Tetrazine-siRNA Conjugation to TCO Labeled Antibody

2 equivalents of methyltetrazine-PEG4-siRNA-PEG5 kDa in pH 7.4 PBS wasadded to the retentate from step-2 and rotated at room temperature for 1hour. The reaction mixture was analyzed by analytical SAX columnchromatography and the antibody-siRNA conjugate was seen along with theunreacted antibody and siRNA.

Step 4: Purification

The crude reaction mixture was purified by AKTA explorer FPLC usinganion exchange chromatography method-1. Fractions containing DAR1 andDAR>2 antibody-siRNA conjugates were separated, concentrated and bufferexchanged with pH 7.4 PBS. Typical DAR>2 lysine conjugate contains70-80% DAR2 and 20-30% DAR3 or higher.

Step-5: Analysis of the Purified Conjugate

The characterization and purity of the isolated conjugate wascharacterized by either mass spec or SDS-PAGE. The purity of theconjugate was assessed by analytical HPLC using anion exchangechromatography method-2.

Scheme-16: Fab-siRNA Conjugate Generation

Step 1: Antibody Digestion with Pepsin

Antibody was buffer exchanged with pH 4.0, 20 mM sodium acetate/aceticacid buffer and made up to 5 mg/ml concentration. Immobilized pepsin(Thermo Scientific, Prod #20343) was added and incubated for 3 hours at37° C. The reaction mixture was filtered using 30 kDa MWCO Amicon spinfilters and pH 7.4 PBS. The retentate was collected and purified usingsize exclusion chromatography to isolate F(ab′)2. The collected F(ab′)2was then reduced by 10 equivalents of TCEP and conjugated withSMCC-C6-siRNA-PEG5 at room temperature in pH 7.4 PBS. Analysis ofreaction mixture on SAX chromatography showed Fab-siRNA conjugate alongwith unreacted Fab and siRNA-PEG. See FIG. 103.

Step 2: Purification

The crude reaction mixture was purified by AKTA explorer FPLC usinganion exchange chromatography method-1. Fractions containing DAR1 andDAR2 Fab-siRNA conjugates were separated, concentrated and bufferexchanged with pH 7.4 PBS.

Step-3: Analysis of the Purified Conjugate

The characterization and purity of the isolated conjugate was assessedby SDS-PAGE and analytical HPLC using anion exchange chromatographymethod-2.

Purification and analytical Methods

Anion Exchange Chromatography Method-1.

Column: Tosoh Bioscience, TSKGel SuperQ-5PW, 21.5 mm ID×15 cm, 13 um

Solvent A: 20 mM TRIS buffer, pH 8.0; Solvent B: 20 mM TRIS, 1.5 M NaCl,pH 8.0; Flow Rate: 6.0 ml/min

Gradient:

a. % A % B Column Volume b. 100 0 1.00 c. 60 40 18.00 d. 40 60 2.00 e.40 60 5.00 f. 0 100 2.00 g. 100 0 2.00

Anion Exchange Chromatography Method-2

Column: Thermo Scientific, ProPac™ SAX-10, Bio LC™, 4×250 mm

Solvent A: 80% 10 mM TRIS pH 8, 20% ethanol; Solvent B: 80% 10 mM TRISpH 8, 20% ethanol, 1.5 M NaCl; Flow Rate: 1.0 ml/min

Gradient:

a. Time % A % B b. 0.0 90 10 c. 3.00 90 10 d. 11.00 40 60 e. 13.00 40 60f. 15.00 90 10 g. 20.00 90 10

Anion Exchange Chromatography Method-3

Column: Thermo Scientific, ProPac™ SAX-10, Bio LC™, 4×250 mm

Solvent A: 80% 10 mM TRIS pH 8, 20% ethanol; Solvent B: 80% 10 mM TRISpH 8, 20% ethanol, 1.5 M NaCl

Flow Rate: 0.75 ml/min

Gradient:

a. Time % A % B b. 0.0 90 10 c. 3.00 90 10 d. 11.00 40 60 e. 23.00 40 60f. 25.00 90 10 g. 30.00 90 10

Size Exclusion Chromatography Method-1

Column: TOSOH Biosciences, TSKgelG3000SW XL, 7.8×300 mm, 5 μM

Mobile phase: 150 mM phosphate buffer

Flow Rate: 1.0 ml/min for 20 mins

siRNA synthesis

All siRNA single strands were fully assembled on solid phase usingstandard phospharamidite chemistry and purified over HPLC. Purifiedsingle strands were duplexed to get the double stranded siRNA.

Each siRNA passenger strand contains two conjugation handles, C6-NH₂ andC6-SH, one at each end of the strand. The passenger strand with C6-NH₂handle at 5′ end contains C6-SH at its 3′ end and the strand thatcontains C6-NH₂ handle at 3′ end contains C6-SH at its 5′ end. Bothconjugation handles are connected to siRNA passenger strand via invertedabasic phosphodiester or phosphorothioate.

A representative structure of siRNA with C6-NH₂ conjugation handle atthe 5′ end and C6-SH at 3′end of the passenger strand is shown in FIG.104.

ASC Architectures Described in Examples 10-41

ASC Architecture-1: Antibody-Lys-SMCC-S-3′-Passenger strand. Thisconjugate (see FIG. 105) was generated by antibody lysine-SMCCconjugation to thiol at the 3′ end of passenger strand.

ASC Architecture-2: Antibody-Cys-SMCC-3′-Passenger strand. Thisconjugate (see FIG. 106) was generated by antibody inter-chain cysteineconjugation to SMCC at the 3′ end of passenger strand.

ASC Architecture-3: Antibody-Lys-SMCC-S-5′-passenger strand. Thisconjugate (see FIG. 107) was generated by antibody lysine-SMCCconjugation to C6-thiol at the 5′ end of passenger strand.

ASC Architecture-4: Antibody-Cys-SMCC-5′-passenger strand. Thisconjugate (see FIG. 108) was generated by antibody inter-chain cysteineconjugation to SMCC at the 5′ end of passenger strand.

ASC Architecture-5: Antibody-Lys-PEG-5′-passenger strand. This conjugate(see FIG. 109) was generated by antibody PEG-TCO conjugation totetrazine at the 5′ end of passenger strand.

ASC Architecture-6: Antibody-Lys-PEG-5′-passenger strand. This conjugate(see FIG. 110) was generated by antibody PEG-TCO conjugation totetrazine at the 5′ end of passenger strand.

ASC Architecture-7: Antibody-Cys-PEG-5′-passenger strand withoutinverted abasic at 5′ end. This conjugate (see FIG. 111) was generatedusing procedure similar to architecture-2. The antibody was conjugateddirectly to the amine on passenger strand 5′ end sugar.

Zalutumumab (EGFR-Ab)

Zalutumumab is a fully human IgG1κ monoclonal antibody directed againstthe human epidermal growth factor receptor (EGFR). It is produced in theChinese Hamster Ovary cell line DJT33, which has been derived from theCHO cell line CHO-K1SV by transfection with a GS vector carrying theantibody genes derived from a human anti-EGFR antibody producinghybridoma cell line (2F8). Standard mammalian cell culture andpurification technologies are employed in the manufacturing ofzalutumumab.

The theoretical molecular weight (MW) of zalutumumab without glycans is146.6 kDa. The experimental MW of the major glycosylated isoform of theantibody is 149 kDa as determined by mass spectrometry. Using SDS-PAGEunder reducing conditions the MW of the light chain was found to beapproximately 25 kDa and the MW of the heavy chain to be approximately50 kDa. The heavy chains are connected to each other by two inter-chaindisulfide bonds, and one light chain is attached to each heavy chain bya single inter-chain disulfide bond. The light chain has two intra-chaindisulfide bonds and the heavy chain has four intra-chain disulfidebonds. The antibody is N-linked glycosylated at Asn305 of the heavychain with glycans composed of N-acetyl-glucosamine, mannose, fucose andgalactose. The predominant glycans present are fucosylated bi-antennarystructures containing zero or one terminal galactose residue. Thecharged isoform pattern of the IgG1K antibody has been investigatedusing imaged capillary IEF, agarose IEF and analytical cation exchangeHPLC. Multiple charged isoforms are found, with the main isoform havingan isoelectric point of approximately 8.7.

The major mechanism of action of zalutumumab is a concentrationdependent inhibition of EGF-induced EGFR phosphorylation in A431 cancercells. Additionally, induction of antibody-dependent cell-mediatedcytotoxicity (ADCC) at low antibody concentrations has been observed inpre-clinical cellular in vitro studies.

Panitumumab (EGFR2-Ab)

Panitumumab is a clinically approved, fully human IgG2 subclassmonoclonal antibody specific to the epidermal growth factor receptor(EGFR). Panitumumab has two gamma heavy chains and two kappa lightchains. Glycosylated panitumumab has a total molecular weight ofapproximately 147 kDa. Panitumumab is expressed as a glycoprotein with asingle consensus N-linked glycosylation site located on the heavy chain.Panitumumab is produced from Chinese Hamster Ovary (CHO) cells andpurified by a series of chromatography steps, viral inactivation step,viral filtration step and ultrafiltration/diafiltration steps.

Panitumumab acts as a competitive antagonist at the ligand binding siteof EGFR to inhibit binding and signaling mediated by EGF andtransforming growth factor α, the natural ligands for this receptor. Theaffinity of binding panitumumab to the EGFR was determined be 3.5 and5.7×10⁻¹²M in recombinant EGFR using BIAcore methods. Inhibition ofbinding of EGF was shown in A431 cells, a human epidermal carcinoma cellline that expresses EGFR. Intracellular acidification, phosphorylationand internalization of the EGFR were blocked in a dose-dependent mannerby panitumumab in A431 cells. Panitumumab was also shown to inhibit cellgrowth in vitro and in vivo in the same cell line.

Herceptin (EGFR3-Ab)

Herceptin is a clinically approved, humanized IgG1 subclass monoclonalantibody specific to the epidermal growth factor receptor2 (EGFR2) alsoknown as Her2. Herceptin has human Fc γ1 isotype along with kappa lightchains.

PSMA-Ab

PSMA-Ab is a humanized IgG1 subclass monoclonal antibody specific toprostate specific membrane antigen (PSMA).

ASGR1-Ab

ASGR mAb-Sino103 is a rabbit IgG monoclonal antibody that binds mouseasialoglycoprotein receptor1 (ASGPR1). It is supplied by SinoBiologicals Inc. (Cat #50083-R103).

ASGR2-Ab

ASGR mAb-R&D is a rat IgG2A subclass monoclonal antibody that bindsmouse asialoglycoprotein receptor1 (ASGPR1). It is purified by protein Aor G from hybridoma culture supernatant and supplied by R&D Systems (Cat# MAB2755)

siRNA-TriGalNAc Conjugate

The siRNA triGalNAc conjugate was synthesized using Lys-Lys dipeptide.Protected triGalNAc was coupled with dipeptide PEG linker and purified.After the removal of carboxylic acid protection group on thetriGalNAc-dipeptide was conjugated to the 5′ end of siRNA passengerstrand.

Example 10: 2016-PK-163-LNCap

siRNA Design and Synthesis

EFGR: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against human EGFR. The sequence ofthe guide/antisense strand was complementary to the gene sequencestarting a base position 333 for the human mRNA transcript for EGFR(ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base, sugar and phosphatemodifications were used to optimize the potency of the duplex and reduceimmunogenicity. All siRNA single strands were fully assembled on solidphase using standard phospharamidite chemistry and purified over HPLC.Purified single strands were duplexed to obtain the double strandedsiRNA. The passenger strand contained two conjugation handles, a C6-NH2at the 5′ end and a C6-SH at the 3′ end. Both conjugation handles wereconnected to siRNA passenger strand via phosphorothioate-invertedabasic-phosphorothioate linker, see Example 9 for the chemicalstructure.

Negative control siRNA sequence (scramble): A published (Burke et al.(2014) Pharm. Res., 31(12):3445-60) 21mer duplex with 19 bases ofcomplementarity and 3′ dinucleotide overhangs was used. The sequence (5′to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ IDNO: 2116). Base, sugar and phosphate modifications were used to reduceimmunogenicity and were comparable to those used in the active siRNA.All siRNA single strands were fully assembled on solid phase usingstandard phospharamidite chemistry and purified over HPLC. Purifiedsingle strands were duplexed to get the double stranded siRNA. Thepassenger strand contained two conjugation handles, a C6-NH₂ at the 5′end and a C6-SH at the 3′ end. Both conjugation handles were connectedto siRNA passenger strand via a phosphodiester-invertedabasic-phosphodiester linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

The AXBYC conjugate used in groups 3-4 were made and purified as a DAR1(n=1) using ASC architecture-4, as described in Example 9. The AXB andAXCYB conjugates were made as described in Example 9.

In Vivo Study Design

Groups (n=5) of female SCID SHO mice bearing subcutaneous flank LNCaPtumors 100-350 mm³ in volume were treated with one intravenous (i.v.)tail vein injection of siRNA conjugate, while control groups (n=5) ofthe same mice received one i.v. injection of PBS as a vehicle control.Treatment groups 1-6 were dosed at 1.0 or 0.5 mg/kg (based on the weightof siRNA) as per the study design below. All groups (treatments andcontrols) were administered a dose volume of 5 mL/kg. Mice weresacrificed by CO₂ asphyxiation at 96 hours post-dose. Table 22 describesthe study design in more detail. 50 mg pieces of tumor and liver, werecollected and snap-frozen in liquid nitrogen. mRNA knockdown in targettissue was determined using a comparative qPCR assay as described inExample 2. Total RNA was extracted from the tissue, reverse transcribedand mRNA levels were quantified using TaqMan qPCR, using theappropriately designed primers and probes. PPIB (housekeeping gene) wasused as an internal RNA loading control, results were calculated by thecomparative Ct method, where the difference between the target gene Ctvalue and the PPIB Ct value (ΔCt) is calculated and then furthernormalized relative to the PBS control group by taking a seconddifference (ΔΔCt). Quantitation of tissue siRNA concentrations weredetermined using a stem-loop qPCR assay as described in Example 2. Theantisense strand of the siRNA was reverse transcribed using a TaqManMicroRNA reverse transcription kit using a sequence-specific stem-loopRT primer. The cDNA from the RT step was then utilized for real-time PCRand Ct values were transformed into plasma or tissue concentrationsusing the linear equations derived from the standard curves.

TABLE 22 siRNA Dose Harvest Dose Volume # of Time Group Test Article N(mg/kg) ROA (mL/kg) Doses (h) 1 PSMA-Ab(Cys)-EGFR (n = 1) 5 1 IV 5.0 196 2 PSMA-Ab(Cys)-EGFR (n = 1) 5 0.5 IV 5.0 1 96 3PSMA-Ab(Cys)-EGFR-PEG5k (n = 1) 5 1 IV 5.0 1 96 4PSMA-Ab(Cys)-EGFR-PEG5k (n = 1) 5 0.5 IV 5.0 1 96 5PSMA-Ab(Cys)-PEG5k-EGFR (n = 1) 5 1 IV 5.0 1 96 6PSMA-Ab(Cys)-PEG5k-EGFR (n = 1) 5 0.5 IV 5.0 1 96 7PSMA-Ab(Cys)-scramble-PEG5k 5 1 IV 5.0 1 96 (n = 1) 8 PBS Control 5 — IV5.0 1 96 Total # of Animals: 40 SCID SHO mice with LNCaP tumors

The orientation of the siRNA and PEG relative to the PSMA-Ab wasexplored in an in vivo mouse tumor model. As illustrated in FIG. 50A,having the siRNA in between the PSMA-Ab and the PEG5k(PSMA-Ab(Cys)-EGFR-PEG5k or the AXBYC format) resulted in higher levelsof EGFR mRNA knockdown in the tumor relative to the alternativeconjugate where PEG5k is in between the PSMA-Ab and the siRNA(PSMA-Ab(Cys)-PEG5k-EGFR or AXCYB format). This approach (AXBYC) alsoresulted in higher levels of EGFR mRNA knockdown in the tumor relativeto the conjugate without PEG5K (PSMA-Ab(Cys)-EGFR or AXB format).

The orientation of the siRNA and PEG relative to the PSMA-Ab was alsoexplored relative to the tissue PK profiles. Tissue concentrations weremeasured pmol/g and then converted to pmol/mL by assuming the density oftissue equals 1 g/mL (a concentration of 1 nM=1 nmol/L=1 pmol/mL=1pmol/g tissue). As illustrated in FIG. 50B, having the siRNA in betweenthe PSMA-Ab and the PEG5k (AXBYC) resulted in higher levels of siRNAdelivery to the tumor relative to the alternative conjugate where PEG5kis in between the PSMA-Ab and the siRNA (AXCYB). This approach (AXBYC)resulted in higher levels of EGFR siRNA delivery to the tumor relativeto the conjugate without PEG5K (AXB).

In a mouse LNCaP subcutaneous xenograph model, it was demonstrated thatthe AXBYC format for the antibody siRNA conjugate resulted in higherlevels of siRNA accumulation in the tumor tissue and a greater magnitudeof EGFR mRNA knockdown, relative to the AXCYB and AXB formats. The LNCaptumor expresses human PSMA, resulting in tumor tissue specificaccumulation of the PSMA targeted siRNA conjugates after i.v.administration, receptor mediate uptake and siRNA facilitated knockdownof the target gene.

Example 11: 2016-PK-202-LNCap

siRNA Design and Synthesis

EFGR: A 21 mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against human EGFR. The sequence ofthe guide/antisense strand was complementary to the gene sequencestarting a base position 333 for the human mRNA transcript for EGFR(ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base, sugar and phosphatemodifications were used to optimize the potency of the duplex and reduceimmunogenicity. All siRNA single strands were fully assembled on solidphase using standard phospharamidite chemistry and purified over HPLC.Purified single strands were duplexed to get the double stranded siRNA.The passenger strand contained two conjugation handles, a C6-NH2 at the5′ end and a C6-SH at the 3′ end. Both conjugation handles wereconnected to siRNA passenger strand via phosphorothioate-invertedabasic-phosphorothioate linker, see Example 9 for the chemicalstructure.

Negative control siRNA sequence (scramble): A published (Burke et al.(2014) Pharm. Res., 31(12):3445-60) 21mer duplex with 19 bases ofcomplementarity and 3′ dinucleotide overhangs was used. The sequence (5′to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ IDNO: 2116). Base, sugar and phosphate modifications were used to reduceimmunogenicity and were comparable to those used in the active siRNA.All siRNA single strands were fully assembled on solid phase usingstandard phospharamidite chemistry and purified over HPLC. Purifiedsingle strands were duplexed to get the double stranded siRNA. Thepassenger strand contained two conjugation handles, a C6-NH2 at the 5′end and a C6-SH at the 3′ end. Both conjugation handles were connectedto siRNA passenger strand via a phosphodiester-invertedabasic-phosphodiester linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

The AXBYC conjugate used in groups 3-5 and 7 was made and purified as aDAR1 (n=1) using ASC architecture-4, as described in Example 9. The AXB(groups 1-2) and AXCYB (group 6) conjugates were made as described inExample 9.

In Vivo Study Design

Groups (n=5) of female SCID SHO mice bearing subcutaneous flank LNCaPtumors 100-350 mm³ in volume were treated with one intravenous (i.v.)tail vein injection of siRNA conjugate, while control groups (n=5) ofthe same mice received one i.v. injection of PBS as a vehicle control.Treatment groups 1-6 were dosed at 1.0 or 0.5 mg/kg (based on the weightof siRNA) as per the study design below. All groups (treatments andcontrols) were administered a dose volume of 5 mL/kg. Mice weresacrificed by CO₂ asphyxiation at 96 hours post-dose. Table 23 describesthe study design in more detail. 50 mg pieces of tumor and liver, werecollected and snap-frozen in liquid nitrogen. mRNA knockdown in targettissue was determined using a comparative qPCR assay as described inExample 2. Total RNA was extracted from the tissue, reverse transcribedand mRNA levels were quantified using TaqMan qPCR, using theappropriately designed primers and probes. PPIB (housekeeping gene) wasused as an internal RNA loading control, results were calculated by thecomparative Ct method, where the difference between the target gene Ctvalue and the PPIB Ct value (ΔCt) is calculated and then furthernormalized relative to the PBS control group by taking a seconddifference (ΔΔCt). Quantitation of tissue siRNA concentrations weredetermined using a stem-loop qPCR assay as described in Example 2. Theantisense strand of the siRNA was reverse transcribed using a TaqManMicroRNA reverse transcription kit using a sequence-specific stem-loopRT primer. The cDNA from the RT step was then utilized for real-time PCRand Ct values were transformed into plasma or tissue concentrationsusing the linear equations derived from the standard curves.

TABLE 23 siRNA Dose Harvest Dose Volume # of Time Group Test Article N(mg/kg) ROA (mL/kg) Doses (h) 1 PSMA-Ab(Cys)-EGFR (n = 1) 5 1 IV 5.0 196 2 PSMA-Ab(Cys)-EGFR (n = 1) 5 0.5 IV 5.0 1 96 3PSMA-Ab(Cys)-EGFR-PEG5k (n = 1) 5 1 IV 5.0 1 96 4PSMA-Ab(Cys)-EGFR-PEG5k (n = 1) 5 0.5 IV 5.0 1 96 5PSMA-Ab(Cys)-EGFR-PEG5k (n = 1) 5 0.25 IV 5.0 1 96 6PSMA-Ab(Cys)-PEG5k-EGFR (n = 1) 5 0.5 IV 5.0 1 96 7PSMA-Ab(Cys)-scramble-PEG5k (n = 1) 5 1 IV 5.0 1 96 8 PBS Control 5 — IV5.0 1 96 Total # of Animals: 40 SCID SHO mice with LNCaP tumors

The orientation of the siRNA and PEG relative to the PSMA-Ab was alsoexplored in an in vivo mouse tumor model. As illustrated in FIG. 51A,having the siRNA in between the PSMA-Ab and the PEG5k(PSMA-Ab(Cys)-EGFR-PEG5k or AXBYC format)) resulted in higher levels ofEGFR mRNA knockdown in the tumor relative to the alternative conjugatewhere PEG5k is in between the PSMA-Ab and the siRNA(PSMA-Ab(Cys)-PEG5k-EGFR or AXCYB format). This approach (AXBYC) alsoresulted in higher levels of EGFR mRNA knockdown in the tumor relativeto the conjugate without PEG5K (PSMA-Ab(Cys)-EGFR or AXB format).

The orientation of the siRNA and PEG relative to the PSMA-Ab was alsoexplored relative to the tissue PK profiles. Tissue concentrations weremeasured pmol/g and then converted to pmol/mL by assuming the density oftissue equals 1 g/mL (a concentration of 1 nM=1 nmol/L=1 pmol/mL=1pmol/g tissue). As illustrated in FIG. 51B, having the siRNA in betweenthe PSMA-Ab and the PEG5k (PSMA-Ab(Cys)-EGFR-PEG5k or AXBYC) resulted inhigher levels of siRNA delivery to the tumor relative to the alternativeconjugate where PEG5k is in between the PSMA-Ab and the siRNA(PSMA-Ab(Cys)-PEG5k-EGFR or AXCYB). This approach (AXBYC) also resultedin higher levels of EGFR siRNA delivery to the tumor relative to theconjugate without PEGSK (PSMA-Ab(Cys)-EGFR or AXB).

In a mouse LNCaP subcutaneous xenograph model, it was demonstrated thatthe AXBYC format for the antibody siRNA conjugate results in higherlevels of siRNA accumulation in the tumor tissue and a greater magnitudeof EGFR mRNA knockdown, relative to the AXCYB and AXB formats. The LNCaptumor expresses human PSMA, resulting in tumor tissue specificaccumulation of the PSMA targeted siRNA conjugates after i.v.administration, receptor mediate uptake and siRNA facilitated knockdownof the target gene.

Example 12: 2016-PK-219-WT

siRNA Design and Synthesis

EFGR: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against human EGFR. The sequence ofthe guide/antisense strand was complementary to the gene sequencestarting a base position 333 for the human mRNA transcript for EGFR(ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082)). Base, sugar and phosphatemodifications were used to optimize the potency of the duplex and reduceimmunogenicity. All siRNA single strands were fully assembled on solidphase using standard phospharamidite chemistry and purified over HPLC.Purified single strands were duplexed to get the double stranded siRNA.The passenger strand contained two conjugation handles, a C6-NH2 at the5′ end and a C6-SH at the 3′ end. Both conjugation handles wereconnected to siRNA passenger strand via phosphorothioate-invertedabasic-phosphorothioate linker, see Example 9 for the chemicalstructure.

Negative control siRNA sequence (scramble): A published (Burke et al.(2014) Pharm. Res., 31(12):3445-60) 21mer duplex with 19 bases ofcomplementarity and 3′ dinucleotide overhangs was used. The sequence (5′to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ IDNO: 2116). Base, sugar and phosphate modifications were used to reduceimmunogenicity and were comparable to those used in the active siRNA.All siRNA single strands were fully assembled on solid phase usingstandard phospharamidite chemistry and purified over HPLC. Purifiedsingle strands were duplexed to get the double stranded siRNA. Thepassenger strand contained two conjugation handles, a C6-NH2 at the 5′end and a C6-SH at the 3′ end. Both conjugation handles were connectedto siRNA passenger strand via a phosphodiester-invertedabasic-phosphodiester linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

The AXBYC conjugate used in groups 4-6 was made and purified as a DAR1(n=1) using ASC architecture-4, as described in Example 9. The AXB(groups 1-3) and AXCYB (groups 7-9) and BYC (groups 10-12) conjugateswere made as described in Example 9.

In Vivo Study Design

Groups (n=4) of wild-type female CD-1 mice were treated with oneintravenous (i.v.) tail vein injections of siRNA conjugates. Treatmentgroups received 0.5 mg/kg (based on the weight of siRNA) and all groupswere administered a dose volume of 5.0 mL/kg. Table 24 illustrates thestudy design in more detail. Non-terminal blood samples were collectedat 5, 30, and 180 minutes post-dose via puncture of the retro-orbitalplexus and centrifuged to generate plasma for PK analysis. Mice weresacrificed by CO₂ asphyxiation at 24, 96, or 168 h post-dose. Terminalblood samples were collected via cardiac puncture and processed togenerate plasma for PK analysis. Quantitation of plasma siRNAconcentrations were determined using a stem-loop qPCR assay as describedin Example 2. The antisense strand of the siRNA was reverse transcribedusing a TaqMan MicroRNA reverse transcription kit using asequence-specific stem-loop RT primer. The cDNA from the RT step wasthen utilized for real-time PCR and Ct values were transformed intoplasma or tissue concentrations using the linear equations derived fromthe standard curves.

TABLE 24 siRNA Survival Terminal Dose # of Dose Bleed Bleed Group TestArticle N (mg/kg) ROA Doses Schedule (min) (h) 1 EGFR-Ab(Cys)-EGFR 4 0.5IV 1 t = 0 5 24 2 (n = 1) 4 0.5 IV 1 t = 0 30 96 3 4 0.5 IV 1 t = 0 180168 4 EGFR-Ab(Cys)-EGFR- 4 0.5 IV 1 t = 0 5 24 5 PEG5k (n = 1) 4 0.5 IV1 t = 0 30 96 6 4 0.5 IV 1 t = 0 180 168 7 EGFR-Ab(Cys)-PEG5k- 4 0.5 IV1 t = 0 5 24 8 EGFR (n = 1) 4 0.5 IV 1 t = 0 30 96 9 4 0.5 IV 1 t = 0180 168 10 EGFR Alone (aka 4 0.5 IV 1 t = 0 5 24 11 EGFR-PEG5k) 4 0.5 IV1 t = 0 30 96 12 4 0.5 IV 1 t = 0 180 168 Total # of Animals: 48 WT miceCD-1

In this in vivo PK experiment the orientation of the siRNA and PEGrelative to the EGFR-Ab was explored to determine the behavior of themAb-siRNA conjugate in plasma. As illustrated in FIG. 52, all themAb-siRNA conjugates (AXB, AXBYC and AXCYB formats) had comparableplasma PK with approximately 10% of the siRNA remaining in the systemiccirculation after 168 hours (7 days), compared to the siRNA-PEGSK (BYCformat) which was rapidly cleared from the plasma.

The AXBYC format for the antibody siRNA conjugate has improved PKproperties relative the siRNA-PEG conjugate (BYC) which was rapidlycleared from the plasma.

Example 13: 2016-PK-199-HCC827

siRNA Design and Synthesis

EFGR: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against human EGFR. The sequence ofthe guide/antisense strand was complementary to the gene sequencestarting a base position 333 for the human mRNA transcript for EGFR(ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082)). Base, sugar and phosphatemodifications were used to optimize the potency of the duplex and reduceimmunogenicity. All siRNA single strands were fully assembled on solidphase using standard phospharamidite chemistry and purified over HPLC.Purified single strands were duplexed to get the double stranded siRNA.

Negative control siRNA sequence (scramble): A published (Burke et al.(2014) Pharm. Res., 31(12):3445-60) 21 mer duplex with 19 bases ofcomplementarity and 3′ dinucleotide overhangs was used. The sequence (5′to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ IDNO: 2116). The same base, sugar and phosphate modifications that wereused for the active EGFR siRNA duplex were used in the negative controlsiRNA. All siRNA single strands were fully assembled on solid phaseusing standard phospharamidite chemistry and purified over HPLC.Purified single strands were duplexed to get the double stranded siRNA.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASCarchitecture-4, as described in Example 9.

In Vivo Study Design

Groups (n=5) of female NCr nu/nu mice bearing subcutaneously (SC) flankHCC827 tumors 100-300 mm³ in volume were treated with one intravenous(i.v.) tail vein injection of siRNA conjugate, while control groups(n=5) of the same mice received one i.v. injection of PBS as a vehiclecontrol. Treatment groups 1-3 and 4-6 were dosed at 1.0, 0.5 or 0.25mg/kg (based on the weight of siRNA) as per the study design below. Asdescribed in Example 9, groups 1-3 contained the same targetingantibody, but groups 4-6 had a different EGFR targeting antibody, whilethe rest of the conjugate components (linker, siRNA and PEG) wereidentical. Group 7 received an antibody conjugate with a negativecontrol siRNA sequence (scramble) as a control for groups 1. All groups(treatments and controls) were administered a dose volume of 5 mL/kg.Mice were sacrificed by CO₂ asphyxiation at 96 hours post-dose. Table 25describes the study design in more detail. 50 mg pieces of tumor andliver, were collected and snap-frozen in liquid nitrogen. mRNA knockdownin target tissue was determined using a comparative qPCR assay asdescribed in Example 2. Total RNA was extracted from the tissue, reversetranscribed and mRNA levels were quantified using TaqMan qPCR, using theappropriately designed primers and probes. PPIB (housekeeping gene) wasused as an internal RNA loading control, results were calculated by thecomparative Ct method, where the difference between the target gene Ctvalue and the PPIB Ct value (ΔCt) is calculated and then furthernormalized relative to the PBS control group by taking a seconddifference (ΔΔCt). Quantitation of tissue siRNA concentrations weredetermined using a stem-loop qPCR assay as described in Example 2. Theantisense strand of the siRNA was reverse transcribed using a TaqManMicroRNA reverse transcription kit using a sequence-specific stem-loopRT primer. The cDNA from the RT step was then utilized for real-time PCRand Ct values were transformed into plasma or tissue concentrationsusing the linear equations derived from the standard curves.

TABLE 25 siRNA Dose Harvest Dose Volume # of Time Group Test Article N(mg/kg) ROA (mL/kg) Doses (h) 1 EGFR-Ab(Cys)-EGFR-PEG5k (n = 1) 5 1 IV5.0 1 96 2 EGFR-Ab(Cys)-EGFR-PEG5k (n = 1) 5 0.5 IV 5.0 1 96 3EGFR-Ab(Cys)-EGFR-PEG5k (n = 1) 5 0.25 IV 5.0 1 96 4EGFR2-Ab(Cys)-EGFR-PEG5k 5 1 IV 5.0 1 96 (n = 1) 5EGFR2-Ab(Cys)-EGFR-PEG5k 5 0.5 IV 5.0 1 96 (n = 1) 6EGFR2-Ab(Cys)-EGFR-PEG5k 5 0.25 IV 5.0 1 96 (n = 1) 7EGFR-Ab(Cys)-scramble-PEG5k 5 1 IV 5.0 1 96 (n = 1) 8 PBS Control 5 — IV5.0 1 96 Total # of Animals: 40 nu/nu mice with HCC82 tumors

siRNA concentrations were determined 96 hours in the tumor and liverafter a single i.v. injection at 1.0, 0.5 and 0.25 mg/kg. Tissueconcentrations were measured pmol/g and then converted to pmol/mL byassuming the density of tissue equals 1 g/mL. In FIG. 53A, aconcentration of 1 nM=1 nmol/L=1 pmol/mL=1 pmol/g tissue. As illustratedin FIG. 53A, both antibody conjugates were capable of delivering higherlevels of siRNA to the tumor relative to the liver, and a dose responsewas observed. The EGFR antibody conjugate was capable of delivering moresiRNA to the tumor tissue, at all the doses tested, relative to theEGFR2 antibody. See FIG. 53B. Both conjugates were capable of EGFR genespecific mRNA knockdown at 96 hours post-administration. The controlconjugate that contained the scrambled siRNA and the PBS vehicle controldid not produce significant EGFR gene specific mRNA knockdown.

As highlighted in FIG. 54, biological activity was demonstrated with theA-X—B—Y—C conjugate with a range of different antibodies and siRNAcargos that are capable of in vivo biological activity in a range ofdifferent tissue targets. In this example, it was demonstrated thattumor specific accumulation of 2 conjugates targeted with two differentEGFR antibodies conjugated to an siRNA designed to down regulate EGFRmRNA. The HCC827 tumor expresses high levels of human EGFR and bothconjugates have a human specific EGFR antibody to target the siRNA,resulting in tumor tissue specific accumulation of the conjugates.Receptor mediate uptake resulted in siRNA mediated knockdown of thetarget gene.

Example 14: 2016-PK-236-HCC827

siRNA Design and Synthesis

EFGR: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against human EGFR. The sequence ofthe guide/antisense strand was complementary to the gene sequencestarting a base position 333 for the human mRNA transcript for EGFR(ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base, sugar and phosphatemodifications were used to optimize the potency of the duplex and reduceimmunogenicity. All siRNA single strands were fully assembled on solidphase using standard phospharamidite chemistry and purified over HPLC.Purified single strands were duplexed to get the double stranded siRNA.The passenger strand contained two conjugation handles, a C6-NH2 at the5′ end and a C6-SH at the 3′ end. Both conjugation handles wereconnected to siRNA passenger strand via phosphorothioate-invertedabasic-phosphorothioate linker, see Example 9 for the chemicalstructure.

Negative control siRNA sequence (scramble): A published (Burke et al.(2014) Pharm. Res., 31(12):3445-60) 21mer duplex with 19 bases ofcomplementarity and 3′ dinucleotide overhangs was used. The sequence (5′to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ IDNO: 2116). Base, sugar and phosphate modifications were used to reduceimmunogenicity and were comparable to those used in the active siRNA.All siRNA single strands were fully assembled on solid phase usingstandard phospharamidite chemistry and purified over HPLC. Purifiedsingle strands were duplexed to get the double stranded siRNA. Thepassenger strand contained two conjugation handles, a C6-NH₂ at the 5′end and a C6-SH at the 3′ end. Both conjugation handles were connectedto siRNA passenger strand via a phosphodiester-invertedabasic-phosphodiester linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASCarchitecture-4, as described in Example 9.

In Vivo Study Design

Groups (n=5) of female NCr nu/nu mice bearing subcutaneously (SC) flankHCC827 tumors 100-300 mm³ in volume were treated with one intravenous(i.v.) tail vein injection of siRNA conjugate, while control group 6(n=5) of the same mice received one i.v. injection of PBS as a vehiclecontrol. Treatment groups 1-3 were dosed at 1.0, 0.5 or 0.25 mg/kg(based on the weight of siRNA), groups 4 and 5 at 1.0 mg/kg, as per thestudy design below. As described in Example 9, groups 1-3 contained thesame targeting antibody, but groups 4 had a different EGFR targetingantibody, while the rest of the conjugate components (linker, siRNA andPEG) were identical. Group 6 received an antibody conjugate with anegative control siRNA sequence (scramble) as a control for groups 5.All groups (treatments and controls) were administered a dose volume of5 mL/kg. Mice were sacrificed by CO₂ asphyxiation at 96 hours post-dose.Table 26 describes the study design in more detail. 50 mg pieces oftumor and liver, were collected and snap-frozen in liquid nitrogen. mRNAknockdown in target tissue was determined using a comparative qPCR assayas described in Example 2. Total RNA was extracted from the tissue,reverse transcribed and mRNA levels were quantified using TaqMan qPCR,using the appropriately designed primers and probes. PPIB (housekeepinggene) was used as an internal RNA loading control, results werecalculated by the comparative Ct method, where the difference betweenthe target gene Ct value and the PPIB Ct value (ΔCt) is calculated andthen further normalized relative to the PBS control group by taking asecond difference (ΔΔCt).

TABLE 26 siRNA Dose Harvest Dose Volume # of Time Group Test Article N(mg/kg) ROA (mL/kg) Doses (h) 1 EGFR3-Ab(Cys)-EGFR- 5 1 IV 5.0 1 96PEG5k (n = 1) 2 EGFR3-Ab(Cys)-EGFR- 5 0.5 IV 5.0 1 96 PEG5k (n = 1) 3EGFR3-Ab(Cys)-EGFR- 5 0.25 IV 5.0 1 96 PEG5k (n = 1) 4EGFR-Ab(Cys)-EGFR- 5 1 IV 5.0 1 96 PEG5k (n = 1) 5EGFR-Ab(Cys)-scramble- 5 1 IV 5.0 1 96 PEG5k (n = 1) 6 PBS Control 5 —IV 5.0 1 96 Total # of Animals: 30 nu/nu mice with HCC827 tumors

In this in vivo PD experiment, it was demonstrated that dose dependentEGFR gene specific mRNA knockdown (FIG. 55) at 96 hour'spost-administration with a third example of an EGFR antibody targetingagent (EGFR3). The control conjugate that contained the scrambled siRNAand the PBS vehicle control did not produce significant EGFR genespecific mRNA knockdown.

As highlighted in FIG. 54, it was demonstrated that biological activitywith the A-X—B—Y—C conjugate with a range of different antibodies andsiRNA cargos that are capable of in vivo biological activity in a rangeof different tissue targets. In this example, it was demonstrated thattumor specific down regulation of EGFR mRNA using a third EGFR antibodytargeting ligand. The HCC827 tumor expresses human EGFR and bothconjugates have a human specific EGFR antibody (EGFR and EGFR3) totarget the siRNA, resulting in tumor tissue specific accumulation of theconjugates. Receptor mediate uptake resulted in siRNA mediated knockdownof the target gene.

Example 15: 2016-PK-234-HCC827

siRNA Design and Synthesis

EFGR: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against human EGFR. The sequence (5′to 3′) of the guide/antisense strand was TCUCGUGCCUUGGCAAACUUU (SEQ IDNO: 2117) and it was design to be complementary to the gene sequencestarting a base position 333 for the human mRNA transcript for EGFR.Base, sugar and phosphate modifications were used to optimize thepotency of the duplex and reduce immunogenicity. All siRNA singlestrands were fully assembled on solid phase using standardphospharamidite chemistry and purified over HPLC. Purified singlestrands were duplexed to get the double stranded siRNA. The passengerstrand contained two conjugation handles, a C6-NH2 at the 5′ end and aC6-SH at the 3′ end. Both conjugation handles were connected to siRNApassenger strand via phosphorothioate-inverted abasic-phosphorothioatelinker, see Example 9 for the chemical structure.

Negative control siRNA sequence (scramble): A published (Burke et al.(2014) Pharm. Res., 31(12):3445-60) 21mer duplex with 19 bases ofcomplementarity and 3′ dinucleotide overhangs was used. The sequence (5′to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ IDNO: 2116). Base, sugar and phosphate modifications were used to reduceimmunogenicity and were comparable to those used in the active siRNA.All siRNA single strands were fully assembled on solid phase usingstandard phospharamidite chemistry and purified over HPLC. Purifiedsingle strands were duplexed to get the double stranded siRNA. Thepassenger strand contained two conjugation handles, a C6-NH₂ at the 5′end and a C6-SH at the 3′ end. Both conjugation handles were connectedto siRNA passenger strand via a phosphodiester-invertedabasic-phosphodiester linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASCarchitecture-4, as described in Example 9.

In Vivo Study Design

Groups (n=5) of female NCr nu/nu mice bearing subcutaneously (SC) flankHCC827 tumors 100-300 mm³ in volume were treated with one intravenous(i.v.) tail vein injection of siRNA conjugate, while control group 10(n=5) of the same mice received one i.v. injection of PBS as a vehiclecontrol. Treatment groups 1-3, 4-6 and 7-9 were dosed at 1.0, 0.5 or0.25 mg/kg (based on the weight of siRNA), as per the study designbelow. As described in Example 9, groups 1-3 contained the sametargeting antibody (EGFR3) but groups 4-9 had a different EGFR targetingantibody, while the rest of the conjugate components (linker, siRNA andPEG) were identical. Group 7-9 received an antibody conjugate with anegative control siRNA sequence (scramble) as a control for groups 1-6.All groups (treatments and controls) were administered a dose volume of5 mL/kg. Mice were sacrificed by CO₂ asphyxiation at 96 hours post-dose.Table 27 describes the study design in more detail. 50 mg pieces oftumor and liver, were collected and snap-frozen in liquid nitrogen. mRNAknockdown in target tissue was determined using a comparative qPCR assayas described in Example 2. Total RNA was extracted from the tissue,reverse transcribed and mRNA levels were quantified using TaqMan qPCR,using the appropriately designed primers and probes. PPIB (housekeepinggene) was used as an internal RNA loading control, results werecalculated by the comparative Ct method, where the difference betweenthe target gene Ct value and the PPIB Ct value (ΔCt) is calculated andthen further normalized relative to the PBS control group by taking asecond difference (MCt). Quantitation of tissue siRNA concentrationswere determined using a stem-loop qPCR assay as described in Example 2.The antisense strand of the siRNA was reverse transcribed using a TaqManMicroRNA reverse transcription kit using a sequence-specific stem-loopRT primer. The cDNA from the RT step was then utilized for real-time PCRand Ct values were transformed into plasma or tissue concentrationsusing the linear equations derived from the standard curves.

TABLE 27 siRNA Dose Harvest Dose Volume # of Time Group Test Article N(mg/kg) ROA (mL/kg) Doses (h) 1 EGFR3-Ab(Cys)-N3′-EGFR-5′S-PEG5k 5 1 IV5.0 1 96 (n = 1) 2 EGFR3-Ab(Cys)-N3′-EGFR-5′S-PEG5k 5 0.5 IV 5.0 1 96 (n= 1) 3 EGFR3-Ab(Cys)-N3′-EGFR-5′S-PEG5k 5 0.25 IV 5.0 1 96 (n = 1) 4EGFR-Ab(Cys)-N5′-EGFR-3′S-PEG5k 5 1 IV 5.0 1 96 (n = 1) 5EGFR-Ab(Cys)-N5′-EGFR-3′S-PEG5k 5 0.5 IV 5.0 1 96 (n = 1) 6EGFR-Ab(Cys)-N5′-EGFR-3′S-PEG5k 5 0.25 IV 5.0 1 96 (n = 1) 7EGFR-Ab(Cys)-N5′-scramble-3′S- 5 1 IV 5.0 1 96 PEG5k (n = 1) 8EGFR-Ab(Cys)-N5′-scramble-3′S- 5 0.5 IV 5.0 1 96 PEG5k (n = 1) 9EGFR-Ab(Cys)-N5′-scramble-3′S- 5 0.25 IV 5.0 1 96 PEG5k (n = 1) 10 PBSControl 5 — IV 5.0 1 96 Total # of Animals: 50 nu/nu mice with HCC827tumors

siRNA concentrations were determined 96 hours in the tumor and liverafter a single i.v. injection at 1.0, 0.5 and 0.25 mg/kg. Tissueconcentrations were measured pmol/g and then converted to pmol/mL byassuming the density of tissue equals 1 g/mL. In FIG. 56A, aconcentration of 1 nM=1 nmol/L=1 pmol/mL=1 pmol/g tissue. As illustratedin FIG. 56A, both antibody conjugates were capable of delivering higherlevels of siRNA to the tumor relative to the liver, and a dose responsewas observed. Both conjugates were capable of EGFR gene specific mRNAknockdown at 96 hours post-administration relative to the scramble andvehicle control. See FIG. 56B.

As highlighted in FIG. 54, it was demonstrated biological activity withthe A-X—B—Y—C conjugate with a range of different antibodies and siRNAcargos that are capable of in vivo biological activity in a range ofdifferent tissue targets. In this example, it was demonstrated tumorspecific accumulation of 2 conjugates targeted with two different EGFRantibodies conjugated to an siRNA designed to down regulate EGFR mRNA.The HCC827 tumor expresses high levels of human EGFR and both conjugateshave a human specific EGFR antibody to target the siRNA, resulting intumor tissue specific accumulation of the conjugates. Receptor mediateuptake resulted in siRNA mediated knockdown of the target gene

Example 16: 2016-PK-237-HCC827

siRNA Design and Synthesis

KRAS: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against human KRAS. The sequence ofthe guide/antisense strand was complementary to the gene sequencestarting a base position 237 for the human mRNA transcript for KRAS(UGAAUUAGCUGUAUCGUCAUU; SEQ ID NO: 2088). Base, sugar and phosphatemodifications were used to optimize the potency of the duplex and reduceimmunogenicity. All siRNA single strands were fully assembled on solidphase using standard phospharamidite chemistry and purified over HPLC.Purified single strands were duplexed to get the double stranded siRNA.The passenger strand contained two conjugation handles, a C6-SH at the3′ end, which was connected to siRNA passenger strand via viaphosphodiester-inverted abasic-phosphorothioate linker. The C6-SH wasconnected through the phosphodiester, see Example 9 for the chemicalstructure. In addition, the 5′ end of the passenger strand had theinverted abasic removed and the antibody was conjugated directly to theamine on passenger strand 5′ end sugar on a T base using a proceduresimilar to architecture 2, see Example 9.

EFGR: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against human EGFR. The sequence ofthe guide/antisense strand was complementary to the gene sequencestarting a base position 333 for the human mRNA transcript for EGFR(ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base, sugar and phosphatemodifications were used to optimize the potency of the duplex and reduceimmunogenicity. All siRNA single strands were fully assembled on solidphase using standard phospharamidite chemistry and purified over HPLC.Purified single strands were duplexed to get the double stranded siRNA.The passenger strand contained two conjugation handles, a C6-NH2 at the5′ end and a C6-SH at the 3′ end. Both conjugation handles wereconnected to siRNA passenger strand via phosphorothioate-invertedabasic-phosphorothioate linker, see Example 9 for the chemicalstructure.

ASC Synthesis and Characterization

Conjugates in groups 1-3 were made and purified as a DAR1 (n=1) usingASC architecture-7, as described in Example 9.

Conjugates in groups 4-6 were made and purified as a DAR1 (n=1) usingASC architecture-4, as described in Example 9.

In Vivo Study Design

Groups (n=5) of female NCr nu/nu mice bearing subcutaneously (SC) flankHCC827 tumors 100-300 mm³ in volume were treated with one intravenous(i.v.) tail vein injection of siRNA conjugate, while control group 7(n=5) of the same mice received one i.v. injection of PBS as a vehiclecontrol. Treatment groups 1-3, 4-6 were dosed at 1.0, 0.5 or 0.25 mg/kg(based on the weight of siRNA), as per the study design below. Asdescribed in Example 9, groups 1-6 contained the same targeting antibody(EGFR) but groups 1-3 had an siRNA designed to downregulate KRAS andgroups 4-6 had an siRNA designed to downregulate EGFR. All groups(treatments and controls) were administered a dose volume of 5 mL/kg.Mice were sacrificed by CO₂ asphyxiation at 96 hours post-dose. Table 28describes the study design in more detail. 50 mg pieces of tumor andliver, were collected and snap-frozen in liquid nitrogen. mRNA knockdownin target tissue was determined using a comparative qPCR assay asdescribed in the methods section. Total RNA was extracted from thetissue, reverse transcribed and mRNA levels were quantified using TaqManqPCR, using the appropriately designed primers and probes. PPIB(housekeeping gene) was used as an internal RNA loading control, resultswere calculated by the comparative Ct method, where the differencebetween the target gene Ct value and the PPIB Ct value (ΔCt) iscalculated and then further normalized relative to the PBS control groupby taking a second difference (ΔΔCt). Quantitation of tissue siRNAconcentrations were determined using a stem-loop qPCR assay as describedin Example 2. The antisense strand of the siRNA was reverse transcribedusing a TaqMan MicroRNA reverse transcription kit using asequence-specific stem-loop RT primer. The cDNA from the RT step wasthen utilized for real-time PCR and Ct values were transformed intoplasma or tissue concentrations using the linear equations derived fromthe standard curves. Plasma concentrations of the antibody component ofthe conjugate were determined using an ELISA assay.

TABLE 28 siRNA Dose Survival Terminal Harvest Dose Volume # of BleedBleed Time Group Test Article N (mg/kg) ROA (mL/kg) Doses (min) (h) (h)1 EGFR-Ab(Cys)-KRAS-PEG5k 5 0.5 IV 5.0 1 0.25 72 72 (n = 1) 2EGFR-Ab(Cys)-KRAS-PEG5k 5 0.5 IV 5.0 1 3 96 96 (n = 1) 3EGFR-Ab(Cys)-KRAS-PEG5k 5 0.5 IV 5.0 1 24 168 168 (n = 1) 4EGFR-Ab(Cys)-EGFR-PEG5k 5 0.5 IV 5.0 1 0.25 72 72 (n = 1) 5EGFR-Ab(Cys)-EGFR-PEG5k 5 0.5 IV 5.0 1 3 96 96 (n = 1) 6EGFR-Ab(Cys)-EGFR-PEG5k 5 0.5 IV 5.0 1 24 168 168 (n = 1) 7 PBS Control5 — IV 5.0 1 — — 96 Total # of Animals: 35 nu/nu mice with HCC827 tumors

siRNA concentrations were determined 96 hours in the tumor and liverafter a single i.v. injection at 1.0, 0.5 and 0.25 mg/kg. Tissueconcentrations were measured pmol/g and then converted to pmol/mL byassuming the density of tissue equals 1 g/mL. In FIG. 57A and FIG. 57B,a concentration of 1 nM=1 nmol/L=1 pmol/mL=1 pmol/g tissue. Asillustrated in FIG. 57A and FIG. 57B, both antibody conjugates werecapable of delivering higher levels of siRNA to the tumor relative tothe liver. The conjugate that contained the siRNA designed todownregulate KRAS was capable of KRAS gene specific mRNA knockdown (FIG.57C) at 96 hours post-administration relative to the conjugate thatcontained the siRNA designed to down regulate EGFR or the PBS vehiclecontrol. Both antibody conjugate constructs had similar PK properties(see FIG. 58A and FIG. 58B) indicating the alternative conjugationstrategy used on the 5′ guide strand for the antibody had no impact onthis biological parameter.

As highlighted in FIG. 54, it was demonstrated biological activity withthe A-X—B—Y—C conjugate with a range of different antibodies and siRNAcargos that are capable of in vivo biological activity in a range ofdifferent tissue targets. In this example it was demonstrated tumorspecific accumulation and siRNA mediated mRNA knockdown of a EGFRantibody conjugated to an siRNA designed to down regulate KRAS mRNA. TheHCC827 tumor expresses high levels of human EGFR and the conjugate has ahuman specific EGFR antibody to target the siRNA, resulting in tumortissue specific accumulation of the conjugates. Receptor mediate uptakeresulted in siRNA mediated knockdown of the KRAS gene.

Example 17: 2016-PK-187-Hep3B

siRNA Design and Synthesis

EFGR: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against human EGFR. The sequence ofthe guide/antisense strand was complementary to the gene sequencestarting a base position 333 for the human mRNA transcript for EGFR(ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base, sugar and phosphatemodifications were used to optimize the potency of the duplex and reduceimmunogenicity. All siRNA single strands were fully assembled on solidphase using standard phospharamidite chemistry and purified over HPLC.Purified single strands were duplexed to get the double stranded siRNA.The passenger strand contained two conjugation handles, a C6-NH2 at the5′ end and a C6-SH at the 3′ end. Both conjugation handles wereconnected to siRNA passenger strand via phosphorothioate-invertedabasic-phosphorothioate linker, see Example 9 for the chemicalstructure.

Negative control siRNA sequence (scramble): A published (Burke et al.(2014) Pharm. Res., 31(12):3445-60) 21mer duplex with 19 bases ofcomplementarity and 3′ dinucleotide overhangs was used. The sequence (5′to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ IDNO: 2116). Base, sugar and phosphate modifications were used to reduceimmunogenicity and were comparable to those used in the active siRNA.All siRNA single strands were fully assembled on solid phase usingstandard phospharamidite chemistry and purified over HPLC. Purifiedsingle strands were duplexed to get the double stranded siRNA. Thepassenger strand contained two conjugation handles, a C6-NH2 at the 5′end and a C6-SH at the 3′ end. Both conjugation handles were connectedto siRNA passenger strand via a phosphodiester-invertedabasic-phosphodiester linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASCarchitecture-4, as described in Example 9.

In Vivo Study Design

Groups (n=5) of female NCr nu/nu mice bearing subcutaneously (SC) flankHep-3B2 1-7 tumors 100-300 mm³ in volume were treated with oneintravenous (i.v.) tail vein injection of siRNA conjugate, while controlgroup 5 (n=5) of the same mice received one i.v. injection of PBS as avehicle control. Treatment groups 1-3 were dosed at 1.0, 0.5 or 0.25mg/kg (based on the weight of siRNA), group 4 (scramble control) wasdosed at 1.0 mg/kg, as per the study design below. Group 4 received anantibody conjugate with a negative control siRNA sequence (scramble) asa control for group 1. All groups (treatments and controls) wereadministered a dose volume of 5 mL/kg. Mice were sacrificed by CO₂asphyxiation at 96 hours post-dose. Table 29 describes the study designin more detail. 50 mg pieces of tumor and liver, were collected andsnap-frozen in liquid nitrogen. mRNA knockdown in target tissue wasdetermined using a comparative qPCR assay as described in Example 2.Total RNA was extracted from the tissue, reverse transcribed and mRNAlevels were quantified using TaqMan qPCR, using the appropriatelydesigned primers and probes. PPIB (housekeeping gene) was used as aninternal RNA loading control, results were calculated by the comparativeCt method, where the difference between the target gene Ct value and thePPIB Ct value (ΔCt) is calculated and then further normalized relativeto the PBS control group by taking a second difference (ΔΔCt).Quantitation of tissue siRNA concentrations were determined using astem-loop qPCR assay as described in Example 2. The antisense strand ofthe siRNA was reverse transcribed using a TaqMan MicroRNA reversetranscription kit using a sequence-specific stem-loop RT primer. ThecDNA from the RT step was then utilized for real-time PCR and Ct valueswere transformed into plasma or tissue concentrations using the linearequations derived from the standard curves.

TABLE 29 siRNA Dose Harvest Dose Volume # of Time Group Test Article N(mg/kg) ROA (mL/kg) Doses (h) 1 EGFR-Ab(Cys)-EGFR-PEG5k (n = 1) 5 1 IV5.0 1 96 2 EGFR-Ab(Cys)-EGFR-PEG5k (n = 1) 5 0.5 IV 5.0 1 96 3EGFR-Ab(Cys)-EGFR-PEG5k (n = 1) 5 0.25 IV 5.0 1 96 4EGFR-Ab(Cys)-scramble-PEG5k 5 1 IV 5.0 1 96 (n = 1) 5 PBS Control 5 — IV5.0 1 96 Total # of Animals: 25 nu/nu mice with Hep3B tumors

siRNA concentrations were determined 96 hours in the tumor and liverafter a single i.v. injection at 1.0, 0.5 and 0.25 mg/kg. Tissueconcentrations were measured pmol/g and then converted to pmol/mL byassuming the density of tissue equals 1 g/mL. In FIG. 59A, aconcentration of 1 nM=1 nmol/L=1 pmol/mL=1 pmol/g tissue. As illustratedin FIG. 59A, the antibody conjugate was capable of delivering siRNA tothe tumor. The conjugate was capable of EGFR gene specific mRNAknockdown (FIG. 59B) at 96 hours post-administration relative to theconjugate that contained the negative control siRNA sequence or the PBSvehicle control.

As highlighted in FIG. 54, it was demonstrated biological activity withthe A-X—B—Y—C conjugate with a range of different antibodies and siRNAcargos that are capable of in vivo biological activity in a range ofdifferent tissue targets. In this example it was demonstrated tumorspecific accumulation and siRNA mediated mRNA knockdown of an EGFRantibody conjugated to an siRNA designed to down regulate EGFR mRNA. TheHep-3B2 1-7 tumor cells express human EGFR and the conjugate has a humanspecific EGFR antibody to target the siRNA, resulting in tumor tissuespecific accumulation of the conjugates. Receptor mediate uptakeresulted in siRNA mediated knockdown of the EGFR gene.

Example 18: 2016-PK-257-WT

siRNA Design and Synthesis

R1442: N5-CTNNB1-3′S

CTNNB1: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against human CTNNB1. The sequenceof the guide/antisense strand was complementary to the gene sequencestarting a base position 1248 for the human mRNA transcript for CTNNB1(UAAUGAGGACCUAUACUUAUU; SEQ ID NO: 2095). Base, sugar and phosphatemodifications were used to optimize the potency of the duplex and reduceimmunogenicity. All siRNA single strands were fully assembled on solidphase using standard phospharamidite chemistry and purified over HPLC.Purified single strands were duplexed to get the double stranded siRNA.The passenger strand contained two conjugation handles, a C6-NH₂ at the5′ end and a C6-SH at the 3′ end. Both conjugation handles wereconnected to the siRNA passenger strand via phosphodiester-invertedabasic-phosphorothioate linker. The C6-NH2 and C6-SH were connectedthrough the phosphodiester, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

The antibody conjugate was made and purified as a DAR1 (n=1) using ASCarchitecture-1, as described in Example 9. The tri-GalNAc-CTNNB1conjugate was made as described in Example 9.

In Vivo Study Design

Groups 1-3 (n=4) of wild-type female CD-1 mice were treated with oneintravenous (i.v.) tail vein injections of siRNA conjugates, the GalNActargeted control was doses by subcutaneous injection. Treatment groups1-3 received doses of 2.0 1.0 and 0.5 mg/kg (based on the weight ofsiRNA) and the GalNAc targeted control conjugate was doses at 2 mg/kg.All groups were administered a dose volume of 5.0 mL/kg. Table 30illustrates the study design in more detail. 50 mg pieces of liver werecollected and snap-frozen in liquid nitrogen. mRNA knockdown in targettissue was determined using a comparative qPCR assay as described inExample 2. Total RNA was extracted from the tissue, reverse transcribedand mRNA levels were quantified using TaqMan qPCR, using theappropriately designed primers and probes. PPIB (housekeeping gene) wasused as an internal RNA loading control, results were calculated by thecomparative Ct method, where the difference between the target gene Ctvalue and the PPIB Ct value (ΔCt) is calculated and then furthernormalized relative to the PBS control group by taking a seconddifference (ΔΔCt).

TABLE 30 siRNA Harvest Dose Dose Volume # of Time Group Test Article N(mg/kg) ROA (mL/kg) Doses (h) 1 ASGR1-Ab(Lys)-CTNNB1- 4 2 IV 5.0 1 96PEG5k (n = 1) 2 ASGR1-Ab(Lys)-CTNNB1- 4 1 IV 5.0 1 96 PEG5k (n = 1) 3ASGR1-Ab(Lys)-CTNNB1- 4 0.5 IV 5.0 1 96 PEG5k (n = 1) 4 3GalNAc-CTNNB1Control 5 2 s.c. 5.0 1 96 5 PBS Control 5 — IV 5.0 1 96 Total # ofAnimals: 22 WT mice (CD-1)

CTNNB1 gene knockdown was determined 96 hours post administration. Asillustrated in FIG. 60, the GalNac-conjugated siRNA was capable of genespecific knockdown after a single s.c injection, as has been welldescribed by others in the field. The same siRNA conjugated to an ASGRantibody was also capable of CTNNB1 gene specific downregulation and ina dose dependent manner.

As highlighted in FIG. 54, it was demonstrated biological activity withthe A-X—B—Y—C conjugate with a range of different antibodies and siRNAcargos that are capable of in vivo biological activity in a range ofdifferent tissue targets. In this example it was demonstrated liverdelivery with an ASGR antibody conjugated to an siRNA designed to downregulate CTNNB1 mRNA. Mouse Liver cells express the asialoglycoproteinreceptor (ASGR) and the conjugate has a mouse specific ASGR antibody totarget the siRNA, resulting in siRNA mediated knockdown of the CTNNB1 inthe liver.

Example 19: 2016-PK-253-WT

siRNA Design and Synthesis

KRAS: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against human KRAS. The sequence ofthe guide/antisense strand was complementary to the gene sequencestarting a base position 237 for the human mRNA transcript for KRAS(UGAAUUAGCUGUAUCGUCAUU; SEQ ID NO: 2088). Base, sugar and phosphatemodifications were used to optimize the potency of the duplex and reduceimmunogenicity. All siRNA single strands were fully assembled on solidphase using standard phospharamidite chemistry and purified over HPLC.Purified single strands were duplexed to get the double stranded siRNA.The passenger strand contained two conjugation handles, a C6-NH₂ at the5′ end and a C6-SH at the 3′ end. Both conjugation handles wereconnected to siRNA passenger strand via phosphodiester-invertedabasic-phosphorothioate linker. The C6-NH₂ and C6-SH were connectedthrough the phosphodiester, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

The antibody conjugate was made and purified as a DAR1 (n=1) using ASCarchitecture-1, as described in Example 9. The tri-GalNAc-CTNNB1conjugate was made as described in Example 9.

In Vivo Study Design

Groups 1-3 (n=4) of wild-type female CD-1 mice were treated with oneintravenous (i.v.) tail vein injections of siRNA conjugates, the GalNActargeted control was doses by subcutaneous injection. Treatment groups1-3 received doses of 2.0 1.0 and 0.5 mg/kg (based on the weight ofsiRNA) and the GalNAc targeted control conjugate was doses at 2 mg/kg.All groups were administered a dose volume of 5.0 mL/kg. Table 31illustrates the study design in more detail. 50 mg pieces of liver werecollected and snap-frozen in liquid nitrogen. mRNA knockdown in targettissue was determined using a comparative qPCR assay as described inExample 2. Total RNA was extracted from the tissue, reverse transcribedand mRNA levels were quantified using TaqMan qPCR, using theappropriately designed primers and probes. PPIB (housekeeping gene) wasused as an internal RNA loading control, results were calculated by thecomparative Ct method, where the difference between the target gene Ctvalue and the PPIB Ct value (ΔCt) is calculated and then furthernormalized relative to the PBS control group by taking a seconddifference (ΔΔCt).

TABLE 31 siRNA Harvest Dose Dose Volume # of Time Group Test Article N(mg/kg) ROA (mL/kg) Doses (h) 1 ASGR2-Ab(Lys)-KRAS- 4 2 IV 5.0 1 96PEG5k (n = 1) 2 ASGR2-Ab(Lys)-KRAS- 4 1 IV 5.0 1 96 PEG5k (n = 1) 3ASGR2-Ab(Lys)-KRAS- 4 0.5 IV 5.0 1 96 PEG5k (n = 1) 4 3GalNAc-KRASControl 5 2 s.c. 5.0 1 96 5 PBS Control 5 — IV 5.0 1 96 Total # ofAnimals: 22 WT mice (CD-1)

KRAS gene knockdown was determined 96 hours post administration. Asillustrated in FIG. 61, the GalNac-conjugated siRNA was capable of genespecific knockdown after a single s.c injection, as has been welldescribed by others in the field. The same siRNA conjugated to an ASGRantibody was also capable of KRAS gene specific downregulation and in adose dependent manner.

As highlighted in FIG. 54, it was demonstrated biological activity withthe A-X—B—Y—C conjugate with a range of different antibodies and siRNAcargos that are capable of in vivo biological activity in a range ofdifferent tissue targets. In this example it was demonstrated liverdelivery with an ASGR antibody conjugated to an siRNA designed to downregulate KRAS mRNA. Mouse Liver cells express the asialoglycoproteinreceptor (ASGR) and the conjugate has a mouse specific ASGR antibody totarget the siRNA, resulting in siRNA mediated knockdown of the KRAS inthe liver

Example 20: 2016-PK-129-WT-plasma

siRNA Design and Synthesis

KRAS: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against human KRAS. The sequence ofthe guide/antisense strand was complementary to the gene sequencestarting a base position 237 for the human mRNA transcript for KRAS(UGAAUUAGCUGUAUCGUCAUU; SEQ ID NO: 2088). Base, sugar and phosphatemodifications were used to optimize the potency of the duplex and reduceimmunogenicity. All siRNA single strands were fully assembled on solidphase using standard phospharamidite chemistry and purified over HPLC.Purified single strands were duplexed to get the double stranded siRNA.The passenger strand contained two conjugation handles, a C6-NH2 at the5′ end and a C6-SH at the 3′ end. Both conjugation handles wereconnected to siRNA passenger strand via a phosphodiester-invertedabasic-phosphodiester linker, see Example 9 for the chemical structure.

EFGR: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against human EGFR. The sequence ofthe guide/antisense strand was complementary to the gene sequencestarting a base position 333 for the human mRNA transcript for EGFR(ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base, sugar and phosphatemodifications were used to optimize the potency of the duplex and reduceimmunogenicity. All siRNA single strands were fully assembled on solidphase using standard phospharamidite chemistry and purified over HPLC.Purified single strands were duplexed to get the double stranded siRNA.The passenger strand contained two conjugation handles, a C6-NH2 at the5′ end and a C6-SH at the 3′ end. Both conjugation handles wereconnected to siRNA passenger strand via phosphorothioate-invertedabasic-phosphorothioate linker, see Example 9 for the chemicalstructure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASCarchitecture-1, as described in Example 9.

In Vivo Study Design

Groups (n=3) of wild-type female CD-1 mice were treated with oneintravenous (i.v.) tail vein injections of siRNA conjugates. Treatmentgroups received 0.5 mg/kg (based on the weight of siRNA) and all groupswere administered a dose volume of 5.0 mL/kg. Table 32 illustrates thestudy design in more detail. Non-terminal blood samples were collectedat 5, 30, and 180 minutes post-dose via puncture of the retro-orbitalplexus and centrifuged to generate plasma for PK analysis. Mice weresacrificed by CO₂ asphyxiation at 24, 96, or 168 h post-dose. Terminalblood samples were collected via cardiac puncture and processed togenerate plasma for PK analysis. Quantitation of plasma siRNAconcentrations were determined using a stem-loop qPCR assay as describedin Example 2. The antisense strand of the siRNA was reverse transcribedusing a TaqMan MicroRNA reverse transcription kit using asequence-specific stem-loop RT primer. The cDNA from the RT step wasthen utilized for real-time PCR and Ct values were transformed intoplasma or tissue concentrations using the linear equations derived fromthe standard curves. Plasma concentrations of antibody were determinedusing an ELISA assay.

TABLE 32 Dose Survival Terminal siRNA Dose Volume # of Bleed Bleed GroupTest Article N (mg/kg) ROA (mL/kg) Doses (min) (h) 1 EGFR2-Ab(Lys)- 30.5 IV 5.0 1 5 24 2 KRAS-PEG5k (N = 1) 3 0.5 IV 50 1 30 96 3 3 0.5 IV5.0 1 180 168 4 PSMA-Ab(Lys)- 3 0.5 IV 5.0 1 5 24 5 EGFR-PEG5k (N = 1) 30.5 IV 5.0 1 30 96 6 3 0.5 IV 5.0 1 180 168 Total # of Animals: 18 WTmice CD-1

In this in vivo PK experiment the plasma clearance of two differentconjugates was explored. As illustrated in FIG. 62, both the mAb-siRNAconjugates had comparable plasma PK when comparing the plasma levels ofthe siRNA (KRAS vs EGFR) or the antibody (EGFR2 vs PSMA).

As highlighted in FIG. 54, it was demonstrated biological activity withthe A-X—B—Y—C conjugate with a range of different antibodies and siRNAcargos that are capable of in vivo biological activity in a range ofdifferent tissue targets. In this example it was demonstrated that twodifferent conjugates with different antibody targeting ligands anddifferent siRNA cargos have comparable plasma PK properties.

Example 21: 2016-PK-123-LNCaP

siRNA Design and Synthesis

EFGR: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against human EGFR. The sequence ofthe guide/antisense strand was complementary to the gene sequencestarting a base position 333 for the human mRNA transcript for EGFR(ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base, sugar and phosphatemodifications were used to optimize the potency of the duplex and reduceimmunogenicity. All siRNA single strands were fully assembled on solidphase using standard phospharamidite chemistry and purified over HPLC.Purified single strands were duplexed to get the double stranded siRNA.The passenger strand contained two conjugation handles, a C6-NH2 at the5′ end and a C6-SH at the 3′ end. Both conjugation handles wereconnected to siRNA passenger strand via phosphorothioate-invertedabasic-phosphorothioate linker, see Example 9 for the chemicalstructure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) or DAR2 (n=2)using ASC architecture-1, as described in Example 9.

In Vivo Study Design

Groups (n=5) of female SCID SHO mice bearing subcutaneous flank LNCaPtumors 100-350 mm³ in volume were treated with one intravenous (i.v.)tail vein injection of siRNA conjugate, while control groups (n=5) ofthe same mice received one i.v. injection of PBS as a vehicle control.Treatment groups were dosed as per the study design in Table 33. Allgroups (treatments and controls) were administered a dose volume of 5.71mL/kg. Mice were sacrificed by CO₂ asphyxiation at 72 hours post-dose.50 mg pieces of tumor and liver, were collected and snap-frozen inliquid nitrogen. mRNA knockdown in target tissue was determined using acomparative qPCR assay as described in Example 2. Total RNA wasextracted from the tissue, reverse transcribed and mRNA levels werequantified using TaqMan qPCR, using the appropriately designed primersand probes. PPIB (housekeeping gene) was used as an internal RNA loadingcontrol, results were calculated by the comparative Ct method, where thedifference between the target gene Ct value and the PPIB Ct value (ΔCt)is calculated and then further normalized relative to the PBS controlgroup by taking a second difference (ΔΔCt). Quantitation of tissue siRNAconcentrations were determined using a stem-loop qPCR assay as describedin Example 2. The antisense strand of the siRNA was reverse transcribedusing a TaqMan MicroRNA reverse transcription kit using asequence-specific stem-loop RT primer. The cDNA from the RT step wasthen utilized for real-time PCR and Ct values were transformed intoplasma or tissue concentrations using the linear equations derived fromthe standard curves.

TABLE 33 Dose Harvest siRNA Dose Volume # of Time Group Test Article N(mg/kg) ROA (mL/kg) Doses (h) 1 PSMA-Ab(Lys)- 5 2 IV 5.71 1 72 2EGFR-PEG5k (n = 1) 5 1 IV 5.71 1 72 3 5 0.5 IV 5.71 1 72 4 PSMA-Ab(Lys)-5 4 IV 5.71 1 72 5 EGFR-PEG5k (n = 2) 5 2 IV 5.71 1 72 6 5 1 IV 5.71 172 7 PSMA-Ab(Lys)- 5 2 IV 5.71 1 72 Scramble-PEG5k (n = 1) 8 EGFR siRNAAlone 5 2 IV 5.71 1 72 9 Vehicle 5 — IV 5.71 1 72 Total # of Animals: 45SCID SHO mice with LNCaP tumors

siRNA concentrations were determined 72 hours in the tumor and liverafter a single i.v. injection, see FIG. 63A. As illustrated in FIG. 63A,the antibody conjugate with a drug to antibody ratio of 1 (n=1) wascapable of delivering siRNA to the tumor in a dose dependent manner, atlevels greater than measured in the liver and produced EGFR genespecific mRNA knockdown relative to the scrambled and PBS controls. Thisis in contrast to the antibody conjugate with a drug to antibody ratioof 2 (n=2), which achieved lower concentrations of siRNA in the tumor atan equivalent dose, liver and tumor concentrations which were of thesame magnitude and a lower levels of EGFR knockdown. The unconjugatedsiRNA had poor tumor and liver accumulation and no measurable EGFR mRNAknockdown. FIG. 63B illustrates relative percentage of EGFR mRNA inLNCaP Tumor.

As highlighted in FIG. 54, it was demonstrated biological activity withthe A-X—B—Y—C conjugate with a range of different antibodies and siRNAcargos that are capable of in vivo biological activity in a range ofdifferent tissue targets. In this example it was demonstrated that theDAR1 conjugate is able to achieve greater siRNA tumor concentrations,relative to the DAR 2 and unconjugated siRNA. In addition, the DAR1conjugate is able to achieve greater levels of siRNA mediate knockdownof EGFR, relative to the DAR 2 and unconjugated siRNA.

Example 22: 2016-PK-258-WT

siRNA Design and Synthesis

HPRT: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against human HPRT. The sequence ofthe guide/antisense strand was AUAAAAUCUACAGUCAUAGUU (SEQ ID NO: 2102)and design to be complementary to the gene sequence starting a baseposition 425 for the human mRNA transcript for HPRT. Base, sugar andphosphate modifications were used to optimize the potency of the duplexand reduce immunogenicity. All siRNA single strands were fully assembledon solid phase using standard phospharamidite chemistry and purifiedover HPLC. Purified single strands were duplexed to get the doublestranded siRNA. The passenger strand contained two conjugation handles,a C6-NH2 at the 5′ end and a C6-SH at the 3′ end. Both conjugationhandles were connected to siRNA passenger strand viaphosphodiester-inverted abasic-phosphorothioate linker. The C6-NH2 andC6-SH were connected through the phosphodiester, see Example 9 for thechemical structure.

Negative control siRNA sequence (scramble): A published (Burke et al.(2014) Pharm. Res., 31(12):3445-60) 21mer duplex with 19 bases ofcomplementarity and 3′ dinucleotide overhangs was used. The sequence (5′to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ IDNO: 2116). The same base, sugar and phosphate modifications that wereused for the active EGFR siRNA duplex were used in the negative controlsiRNA. All siRNA single strands were fully assembled on solid phaseusing standard phospharamidite chemistry and purified over HPLC.Purified single strands were duplexed to get the double stranded siRNA.The passenger strand contained two conjugation handles, a C6-NH₂ at the5′ end and a C6-SH at the 3′ end. Both conjugation handles wereconnected to siRNA passenger strand via a phosphodiester-invertedabasic-phosphodiester linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

Conjugates in groups 1-3 and 7-9 were made and purified as a DAR1 (n=1)using ASC architecture-4, as described in Example 9. Conjugates ingroups 4-6 were made and purified as a DAR1 (n=1) using ASCarchitecture-1, as described in Example 9.

In Vivo Study Design

Groups (n=4) of wild-type female CD-1 mice were treated with oneintravenous (i.v.) tail vein injections of siRNA conjugates, while thecontrol group (n=4) of the same mice received one i.v. injection of PBSas a vehicle control. Table 34 illustrates the study design in moredetail. 50 mg pieces of tissue, were collected and snap-frozen in liquidnitrogen. mRNA knockdown in target tissue was determined using acomparative qPCR assay as described in Example 2. Total RNA wasextracted from the tissue, reverse transcribed and mRNA levels werequantified using TaqMan qPCR, using the appropriately designed primersand probes. PPIB (housekeeping gene) was used as an internal RNA loadingcontrol, results were calculated by the comparative Ct method, where thedifference between the target gene Ct value and the PPIB Ct value (ΔCt)is calculated and then further normalized relative to the PBS controlgroup by taking a second difference (ΔΔCt). Quantitation of tissue siRNAconcentrations were determined using a stem-loop qPCR assay as describedin Example 2. The antisense strand of the siRNA was reverse transcribedusing a TaqMan MicroRNA reverse transcription kit using asequence-specific stem-loop RT primer. The cDNA from the RT step wasthen utilized for real-time PCR and Ct values were transformed intoplasma or tissue concentrations using the linear equations derived fromthe standard curves.

TABLE 34 siRNA Dose Harvest Dose Volume # of Time Group Test Article N(mg/kg) ROA (mL/kg) Doses (h) 1 Anti-B cell Ab(Cys)-HPRT-PEG5k 4 3 IV5.0 1 96 (n = 1) 2 Anti-B cell Ab(Cys)-HPRT-PEG5k 4 1 IV 5.0 1 96 (n= 1) 3 Anti-B cell Ab(Cys)-HPRT-PEG5k 4 0.3 IV 5.0 1 96 (n = 1) 4 Anti-Bcell Ab(Lys)-HPRT-PEG5k 4 3 IV 5.0 1 96 (n = 1) 5 Anti-B cellAb(Lys)-HPRT-PEG5k 4 1 IV 5.0 1 96 (n = 1) 6 Anti-B cellAb(Lys)-HPRT-PEG5k 4 0.3 IV 5.0 1 96 (n = 1) 7 Anti-B cellAb(Cys)-scramble-PEG5k 4 3 IV 5.0 1 96 (n = 1) 8 Anti-B cellAb(Cys)-scramble-PEG5k 4 1 IV 5.0 1 96 (n = 1) 9 Anti-B cellAb(Cys)-scramble-PEG5k 4 0.3 IV 5.0 1 96 (n = 1) 10 PBS Control 4 — IV5.0 1 96 Total # of Animals: 77 WT mice (CD-1)

As illustrated on FIG. 64A-FIG. 64C, after a single i.v. administrationof an ASC dose dependent knockdown of HPRT in heart muscle, gastrocskeletal muscle and liver were measured. There was no measurableknockdown of HPRT in the lung tissue (FIG. 64D). In addition, dosedependent accumulation of the siRNA in all four tissue compartments wasobserved (FIG. 64E). There was no significant difference in thebiological activity (KD and tissue concentration) between the lysine andcysteine conjugates.

As highlighted in FIG. 54, it was demonstrated biological activity withthe A-X—B—Y—C conjugate with a range of different antibodies and siRNAcargos that are capable of in vivo biological activity in a range ofdifferent tissue targets. In this example it was demonstrated that ananti-B cell antibody can be used to target an siRNA to heart muscle,gastroc skeletal muscle and liver and achieve gene specificdownregulation of the reporter gene HPRT. There was no measurabledifference in the biological activity of the ASC constructs when alysine or cysteine conjugation strategy was use to attach to theantibody.

Example 23: 2016-PK-254-WT

siRNA Design and Synthesis

HPRT: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against human HPRT. The sequence ofthe guide/antisense strand was AUAAAAUCUACAGUCAUAGUU (SEQ ID NO: 2102)and design to be complementary to the gene sequence starting a baseposition 425 for the human mRNA transcript for HPRT. Base, sugar andphosphate modifications that are well described in the field of RNAiwere used to optimize the potency of the duplex and reduceimmunogenicity. All siRNA single strands were fully assembled on solidphase using standard phospharamidite chemistry and purified over HPLC.Purified single strands were duplexed to get the double stranded siRNA.The passenger strand contained two conjugation handles, a C6-NH₂ at the5′ end and a C6-SH at the 3′ end. Both conjugation handles wereconnected to siRNA passenger strand via phosphodiester-invertedabasic-phosphorothioate linker. The C6-NH₂ and C6-SH were connectedthrough the phosphodiester, see Example 9 for the chemical structure.

Negative control siRNA sequence (scramble): A published (Burke et al.(2014) Pharm. Res., 31(12):3445-60) 21 mer duplex with 19 bases ofcomplementarity and 3′ dinucleotide overhangs was used. The sequence (5′to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ IDNO: 2116). The same base, sugar and phosphate modifications that wereused for the active EGFR siRNA duplex were used in the negative controlsiRNA. All siRNA single strands were fully assembled on solid phaseusing standard phospharamidite chemistry and purified over HPLC.Purified single strands were duplexed to get the double stranded siRNA.The passenger strand contained two conjugation handles, a C6-NH2 at the5′ end and a C6-SH at the 3′ end. Both conjugation handles wereconnected to siRNA passenger strand via a phosphodiester-invertedabasic-phosphodiester linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASCarchitecture-4, as described in Example 9.

In Vivo Study Design

Groups (n=4) of wild-type female CD-1 mice were treated with oneintravenous (i.v.) tail vein injections of siRNA conjugates, while thecontrol group (n=5) of the same mice received one i.v. injection of PBSas a vehicle control. Table 35 illustrates the study design in moredetail. 50 mg pieces of tissue, were collected and snap-frozen in liquidnitrogen. mRNA knockdown in target tissue was determined using acomparative qPCR assay as described in Example 2. Total RNA wasextracted from the tissue, reverse transcribed and mRNA levels werequantified using TaqMan qPCR, using the appropriately designed primersand probes. PPIB (housekeeping gene) was used as an internal RNA loadingcontrol, results were calculated by the comparative Ct method, where thedifference between the target gene Ct value and the PPIB Ct value (ΔCt)is calculated and then further normalized relative to the PBS controlgroup by taking a second difference (ΔΔCt). Quantitation of tissue siRNAconcentrations were determined using a stem-loop qPCR assay as describedin Example 2. The antisense strand of the siRNA was reverse transcribedusing a TaqMan MicroRNA reverse transcription kit using asequence-specific stem-loop RT primer. The cDNA from the RT step wasthen utilized for real-time PCR and Ct values were transformed intoplasma or tissue concentrations using the linear equations derived fromthe standard curves.

TABLE 35 siRNA Dose Harvest Dose Volume # of Time Group Test Article N(mg/kg) ROA (mL/kg) Doses (h) 1 Anti-B cell Fab(Cys)-HPRT-PEG5k 4 10 IV5.1 1 96 (n = 1) 2 Anti-B cell Fab(Cys)-HPRT-PEG5k 4 3 IV 5.1 1 96 (n= 1) 3 Anti-B cell Fab(Cys)-HPRT-PEG5k 4 1 IV 5.1 1 96 (n = 1) 4 Anti-Bcell Fab(Cys)-scramble-PEG5k 4 10 IV 5.1 1 96 (n = 1) 5 Anti-B cellFab(Cys)-scramble-PEG5k 4 3 IV 5.1 1 96 (n = 1) 6 Anti-B cellFab(Cys)-scramble-PEG5k 4 1 IV 5.1 1 96 (n = 1) 7 PBS Control 5 — IV 5.11 96 Total # of Animals: 29 WT mice (CD-1)

As illustrated on FIG. 65A-FIG. 65C, after a single i.v. administrationof an ASC containing an anti-B cell Fab targeting ligand, dose dependentknockdown of HPRT in heart muscle, gastroc skeletal muscle and liverwere measured. There was no measurable knockdown of HPRT in the lungtissue (FIG. 65D). In addition, dose dependent accumulation of the siRNAin all four tissue compartments was observed (FIG. 65E).

As highlighted in FIG. 54, biological activity was demonstrated with theA-X—B—Y—C conjugate with a range of different antibodies and siRNAcargos that are capable of in vivo biological activity in a range ofdifferent tissue targets. In this example it was demonstrated that ananti-B cell Fab is used to target an siRNA to heart muscle, gastrocskeletal muscle and liver and achieve gene specific downregulation ofthe reporter gene HPRT.

Example 24: 2016-PK-245-WT

siRNA Design and Synthesis

CTNNB1: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against human CTNNB1. The sequenceof the guide/antisense strand was TUUCGAAUCAAUCCAACAGUU (SEQ ID NO:2096), design to target the gene sequence starting a base position 1797for the human mRNA transcript for CTNNB1. Base, sugar and phosphatemodifications were used to optimize the potency of the duplex and reduceimmunogenicity. All siRNA single strands were fully assembled on solidphase using standard phospharamidite chemistry and purified over HPLC.Purified single strands were duplexed to get the double stranded siRNA.The passenger strand contained two conjugation handles, a C6-NH₂ at the5′ end and a C6-SH at the 3′ end. Both conjugation handles wereconnected to siRNA passenger strand via phosphodiester-invertedabasic-phosphorothioate linker. The C6-NH2 and C6-SH were connectedthrough the phosphodiester, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

Conjugates in groups 3-4 were made and purified as a DAR1 (n=1) usingASC architecture-4, as described in Example 9. Conjugates in groups 1-2were made and purified as a DAR1 (n=1) using ASC architecture-1, asdescribed in Example 9.

In Vivo Study Design

Groups (n=4) of wild-type female CD-1 mice were treated with oneintravenous (i.v.) tail vein injections of siRNA conjugates, while thecontrol group (n=5) of the same mice received one i.v. injection of PBSas a vehicle control. Table 36 illustrates the study design in moredetail. 50 mg pieces of tissue, were collected and snap-frozen in liquidnitrogen. mRNA knockdown in target tissue was determined using acomparative qPCR assay as described in Example 2. Total RNA wasextracted from the tissue, reverse transcribed and mRNA levels werequantified using TaqMan qPCR, using the appropriately designed primersand probes. PPIB (housekeeping gene) was used as an internal RNA loadingcontrol, results were calculated by the comparative Ct method, where thedifference between the target gene Ct value and the PPIB Ct value (ΔCt)is calculated and then further normalized relative to the PBS controlgroup by taking a second difference (ΔΔCt). Quantitation of tissue siRNAconcentrations were determined using a stem-loop qPCR assay as describedin Example 2. The antisense strand of the siRNA was reverse transcribedusing a TaqMan MicroRNA reverse transcription kit using asequence-specific stem-loop RT primer. The cDNA from the RT step wasthen utilized for real-time PCR and Ct values were transformed intoplasma or tissue concentrations using the linear equations derived fromthe standard curves.

TABLE 36 siRNA Dose Harvest Dose Volume # of Time Group Test Article N(mg/kg) ROA (mL/kg) Doses (h) 1 Anti-B cell Ab(Lys)-CTNNB1-PEG5k 4 3 IV5.0 1 96 (n = 1) 2 Anti-B cell Ab(Lys)-CTNNB1-PEG5k 4 1 IV 5.0 1 96 (n= 1) 3 Anti-B cell Ab(Cys)-CTNNB1-PEG5k 4 3 IV 5.0 1 96 (n = 1) 4 Anti-Bcell Ab(Cys)-CTNNB1-PEG5k 4 1 IV 5.0 1 96 (n = 1) 5 PBS Control 5 — IV5.0 1 96 Total # of Animals: 21 WT mice (CD-1)

As illustrated on FIG. 66A and FIG. 66B, after a single i.v.administration of an ASC containing an anti-B cell antibody targetingligand (anti-B cell-Ab), HPRT knockdown and dose dependent tissue siRNAaccumulation in heart muscle were elicited. As illustrated on FIG. 66Cand FIG. 66D, after a single i.v. administration of an ASC containing ananti-B cell antibody targeting ligand, HPRT knockdown and dose dependenttissue siRNA accumulation in gastroc skeletal muscle were elicited.There was no significant difference in the biological activity (KD andtissue concentration) between the lysine and cysteine conjugates.

As highlighted in FIG. 54, it was demonstrated biological activity withthe A-X—B—Y—C conjugate with a range of different antibodies and siRNAcargos that are capable of in vivo biological activity in a range ofdifferent tissue targets. In this example, it was demonstrated that ananti-B cell antibody is used to target an siRNA to heart muscle andgastroc skeletal muscle and achieve gene specific downregulation ofCTNNB1 mRNA.

Example 25: 2016-PK-160-LNCaP

siRNA Design and Synthesis

AR: A 21mer duplex with 19 bases of complementarity and 3′ dinucleotideoverhangs was designed against human AR. The sequence of theguide/antisense strand was complementary to the gene sequence starting abase position 2822 for the human mRNA transcript for AR (Guide strandsequence: GAGAGCUCCAUAGUGACACUU; SEQ ID NO: 2108). Base, sugar andphosphate modifications were used to optimize the potency of the duplexand reduce immunogenicity. All siRNA single strands were fully assembledon solid phase using standard phospharamidite chemistry and purifiedover HPLC. Purified single strands were duplexed to get the doublestranded siRNA. The passenger strand contained two conjugation handles,a C6-NH₂ at the 5′ end and a C6-SH at the 3′ end. Both conjugationhandles were connected to siRNA passenger strand viaphosphorothioate-inverted abasic-phosphorothioate linker, see Example 9for the chemical structure.

Negative control siRNA sequence (scramble): A published (Burke et al.(2014) Pharm. Res., 31(12):3445-60) 21mer duplex with 19 bases ofcomplementarity and 3′ dinucleotide overhangs was used. The sequence (5′to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ IDNO: 2116). Base, sugar and phosphate modifications were used to reduceimmunogenicity and were comparable to those used in the active siRNA.All siRNA single strands were fully assembled on solid phase usingstandard phospharamidite chemistry and purified over HPLC. Purifiedsingle strands were duplexed to get the double stranded siRNA. Thepassenger strand contained two conjugation handles, a C6-NH₂ at the 5′end and a C6-SH at the 3′ end. Both conjugation handles were connectedto siRNA passenger strand via a phosphodiester-invertedabasic-phosphodiester linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASCarchitecture-1, as described in Example 9.

In Vivo Study Design

Groups (n=5) of female SCID SHO mice bearing subcutaneous flank LNCaPtumors 100-350 mm³ in volume were treated with one intravenous (i.v.)tail vein injection of siRNA conjugate, while control group (n=5) of thesame mice received one i.v. injection of PBS as a vehicle control. Thetable below describes the study design. Mice were sacrificed by CO₂asphyxiation at 96 hours post-dose. 50 mg pieces of tumor and liver,were collected and snap-frozen in liquid nitrogen. mRNA knockdown intarget tissue was determined using a comparative qPCR assay as describedin Example 2. Total RNA was extracted from the tissue, reversetranscribed and mRNA levels were quantified using TaqMan qPCR, using theappropriately designed primers and probes. PPIB (housekeeping gene) wasused as an internal RNA loading control, results were calculated by thecomparative Ct method, where the difference between the target gene Ctvalue and the PPIB Ct value (ΔCt) is calculated and then furthernormalized relative to the PBS control group by taking a seconddifference (ΔΔCt). Quantitation of tissue siRNA concentrations weredetermined using a stem-loop qPCR assay as described in Example 2. Theantisense strand of the siRNA was reverse transcribed using a TaqManMicroRNA reverse transcription kit using a sequence-specific stem-loopRT primer. The cDNA from the RT step was then utilized for real-time PCRand Ct values were transformed into plasma or tissue concentrationsusing the linear equations derived from the standard curves.

TABLE 37 siRNA Dose Harvest Dose Volume # of Time Group Test Article N(mg/kg) ROA (mL/kg) Doses (h) 1 ANT4044(Lys)-AR-PEG5k (n = 1) 5 1 IV 5.01 96 2 ANT4044(Lys)-AR-PEG5k (n = 1) 5 0.5 IV 5.0 1 96 3ANT4044(Lys)-AR-PE5k (n = 1) 5 0.25 IV 5.0 1 96 4ANT4044(Lys)-scramble-PEG5k 5 1 IV 5.0 1 96 (n = 1) 5 PBS Control 5 — IV5.0 1 96 Total # of Animals: 30 castrated SCID SHO mice with LNCaPtumors

As illustrated in FIG. 67A, after a single i.v. administration of an ASCcontaining a PSMA antibody targeting ligand and siRNA designed todownregulate AR, AR knockdown in the LNCaP tumor tissue at all the dosestested relative to the scrambled control was elicited. As illustratedFIG. 67B, there was measurable accumulation of siRNA in the tumor tissueand at levels higher than those measured in the liver tissue.

As highlighted in FIG. 54, it was demonstrated biological activity withthe A-X—B—Y—C conjugate with a range of different antibodies and siRNAcargos that are capable of in vivo biological activity in a range ofdifferent tissue targets. In this example, it was demonstrated deliveryto an LNCaP prostate tumor with a PSMA antibody conjugated to an siRNAdesigned to down regulate AR mRNA. LNCaP cells express human PSMA oncell surface, the conjugate has a human specific PSMA antibody thatbinds to the antigen and allows internalization of the siRNA, resultingin siRNA mediated knockdown of AR in the tumor tissue.

Example 26: In Vitro Uptake and Knockdown in B Cells

siRNA Design and Synthesis

HPRT: A 21 mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against human HPRT. The sequence ofthe guide/antisense strand was AUAAAAUCUACAGUCAUAGUU (SEQ ID NO: 2102)and design to be complementary to the gene sequence starting a baseposition 425 for the human mRNA transcript for HPRT. Base, sugar andphosphate modifications were used to optimize the potency of the duplexand reduce immunogenicity. All siRNA single strands were fully assembledon solid phase using standard phospharamidite chemistry and purifiedover HPLC. Purified single strands were duplexed to get the doublestranded siRNA. The passenger strand contained two conjugation handles,a C6-NH₂ at the 5′ end and a C6-SH at the 3′ end. Both conjugationhandles were connected to siRNA passenger strand viaphosphodiester-inverted abasic-phosphorothioate linker. The C6-NH₂ andC6-SH were connected through the phosphodiester, see Example 9 for thechemical structure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASCarchitecture-4, as described in Example 9.

In Vitro Study Design

Mouse spleens were harvested and kept in PBS with 100 u/ml penicillinand streptomycin on ice. Spleens were smashed with clean glass slides,cut into small pieces, homogenized with 18G needles, and filtered (70urn nylon membrane). Dead cells were removed with the dead cell removalkit from Milteny biotec (Catalog #130-090101) according to manufacturerinstruction. To isolate mouse B cells, B cell isolation kit Miltenybiotec (Catalog #130-090-862) was used following manufacturerinstruction. Briefly, live spleen cells were resuspended with 200 μl ofMACS buffer per mouse spleen. Non-B cells were depleted withbiotin-conjugated monoclonal antibodies against CD43 (Ly48), CD4, andTer-119, coupled with anti-biotin magnetic microbeads. From one mousespleen, 30 million live B cells can be obtained. To activate isolatedmouse B cells (2×10⁶/ml in 10% FBS RPMI-1640 with 100 u/ml penicillinand streptomycin), a cocktail of 10 μg/ml LPS, 5 μg/ml anti-IgM, 1 μg/mlanti-CD40, 0.05 μg/ml IL-4, and 0.05 μg/ml INFγ was added. After fourhours of activation, ASCs (1 μM to 10 nM) were added to 10⁶ cells perwell in 24 (0.5 ml media) or 12 (1 ml media) well plates. After 48 hoursof ASC treatments, cells were harvested and isolated RNAs were analyzedfor mRNA knockdown.

TABLE 38 Group Test Article 1 Anti-B cell Ab(Cys)-HPRT-PEG5k (n = 1) 2Anti-B cell Ab (Cys)-scramble-PEG5k (n = 1) 3 Anti-B cellFab(Cys)-HPRT-PEG5k (n = 1) 4 Anti-B cell Fab(Cys)-scramble-PEG5k (n= 1) 5 Anti-B cell Ab 6 Vehicle Control

In this in vitro experiment in activated primary mouse B cells, theability of an anti-B cell antibody and Fab ASCs to deliver an siRNAdesign to downregulate Hypoxanthine-guanine phosphoribosyltransferase(HPRT) was measured. As illustrated in FIG. 68A, the Fab conjugate wasable to downregulate HPRT relative to the vehicle or scramble controls.As illustrated in FIG. 68B, the antibody conjugate was able todownregulate HPRT relative to the antibody, vehicle, and scramblecontrols.

As highlighted in FIG. 54, it was demonstrated biological activity withthe A-X—B—Y—C conjugate with a range of different antibodies and siRNAcargos that are capable of in vivo biological activity in a range ofdifferent tissue targets. In this example, it was demonstrated deliveryto an activated mouse B cell with a mouse anti-B cell antibody or anti-Bcell Fab conjugated to an siRNA designed to down regulate HPRT mRNA.Activated mouse B cells recognize and internalize the antibody-siRNAconjugate via surface receptors that recognize the anti-B cell antibody,resulting in siRNA mediated knockdown of HPRT.

Example 27: 2016-PK-249-WT

siRNA Design and Synthesis

EFGR: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against human EGFR. The sequence ofthe guide/antisense strand was complementary to the gene sequencestarting a base position 333 for the human mRNA transcript for EGFR(ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base, sugar and phosphatemodifications were used to optimize the potency of the duplex and reduceimmunogenicity. All siRNA single strands were fully assembled on solidphase using standard phospharamidite chemistry and purified over HPLC.Purified single strands were duplexed to get the double stranded siRNA.The passenger strand contained two conjugation handles, a C6-NH₂ at the5′ end and a C6-SH at the 3′ end. Both conjugation handles wereconnected to siRNA passenger strand via phosphorothioate-invertedabasic-phosphorothioate linker, see Example 9 for the chemicalstructure.

KRAS: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against human EGFR. The sequence ofthe guide/antisense strand was complementary to the gene sequencestarting a base position 237 for the human mRNA transcript for KRAS(UGAAUUAGCUGUAUCGUCAUU; SEQ ID NO: 2088). Base, sugar and phosphatemodifications were used to optimize the potency of the duplex and reduceimmunogenicity. All siRNA single strands were fully assembled on solidphase using standard phospharamidite chemistry and purified over HPLC.Purified single strands were duplexed to get the double stranded siRNA.The passenger strand contained two conjugation handles, a C6-NH₂ at the5′ end and a C6-SH at the 3′ end. Both conjugation handles wereconnected to siRNA passenger strand via phosphorothioate-invertedabasic-phosphorothioate linker, see Example 9 for the chemicalstructure.

ASC Synthesis and Characterization

The conjugate for groups 1-2 were made and purified as a DAR1 (n=1)using ASC architecture-4, as described in Example 9. The conjugate forgroups 3-4 were made and purified as a DAR2 (n=2) using ASCarchitecture-4, as described in Example 9. The conjugate for groups 5-6were made and purified as a DAR1 (n=1) using ASC architecture-5, asdescribed in Example 9. The conjugate for groups 7-8 were made andpurified as a DAR2 (n=2) using ASC architecture-5, as described inExample 9. The conjugate for groups 9-10 were made and purified as aDAR1 (n=1) using ASC architecture-6, as described in Example 9. Theconjugate for groups 11-12 were made and purified as a DAR2 (n=2) usingASC architecture-6, as described in Example 9.

In Vivo Study Design

Groups (n=4) of wild-type female CD-1 mice were treated with oneintravenous (i.v.) tail vein injections of siRNA conjugates (groups1-12) or antibody alone (groups 13-14). Table 39 illustrates the studydesign. Non-terminal blood samples were collected at 0.25, and 3 hourspost-dose via puncture of the retro-orbital plexus and centrifuged togenerate plasma for PK analysis. Mice were sacrificed by CO₂asphyxiation at 24 and 72 hours post-dose. Terminal blood samples werecollected via cardiac puncture and processed to generate plasma for PKanalysis. Quantitation of plasma siRNA concentrations were determinedusing a stem-loop qPCR assay as described in Example 2. The antisensestrand of the siRNA was reverse transcribed using a TaqMan MicroRNAreverse transcription kit using a sequence-specific stem-loop RT primer.The cDNA from the RT step was then utilized for real-time PCR and Ctvalues were transformed into plasma or tissue concentrations using thelinear equations derived from the standard curves. Plasma concentrationsof antibody were determined using an ELISA assay.

TABLE 39 siRNA Dose Survival Terminal Dose Volume # of Bleed Bleed GrTest Article N (mg/kg) (mL/kg) Doses (h) (h) 1 EGFR-Ab(Cys)-EGFR-PEG5k(n = 1) 4 0.5 5.0 1 0.25 24 2 EGFR-Ab(Cys)-EGFR-PEG5k (n = 1) 4 0.5 5.01 3 72 3 EGFR-Ab(Cys)-EGFR-PEG5k (n = 2) 4 0.5 5.0 1 0.25 24 4EGFR-Ab(Cys)-EGFR-PEG5k (n = 2) 4 0.5 5.0 1 3 72 5EGFR-Ab(Lys-DHPz)-KRAS-PEG5k 4 0.5 5.0 1 0.25 24 (n = 1) 6EGFR-Ab(Lys-DHPz)-KRAS-PEG5k 4 0.5 5.0 1 3 72 (n = 1) 7EGFR-Ab(Lys-DHPz)-KRAS-PEG5k 4 0.5 5.0 1 0.25 24 (n = 2) 8EGFR-Ab(Lys-DHPz)-KRAS-PEG5k 4 0.5 5.0 1 3 72 (n = 2) 9EGFR-Ab(Asn297-DHPz)-KRAS- 4 0.125 5.0 1 0.25 24 PEG5k (n = 1) 10EGFR-Ab(Asn297-DHPz)-KRAS- 4 0.125 5.0 1 3 72 PEG5k (n = 1) 11EGFR-Ab(Asn297-DHPz)-KRAS- 4 0.125 5.0 1 0.25 24 PEG5k (n = 2) 12EGFR-Ab(Asn297-DHPz)-KRAS- 4 0.125 5.0 1 3 72 PEG5k (n = 2) 13 EGFR-Ab 40.5 5.0 1 0.25 24 14 EGFR-Ab 4 0.5 5.0 1 3 72 Total # of Animals: 56 WTmice CD-1

In this in vivo PK study it was demonstrated the utility of sitespecific conjugation. As illustrated in FIG. 69A, the DAR1 (n=1) testarticle (group 9) had a comparable siRNA plasma clearance profile to thetwo controls (groups 1 and 5), with approximately 10% of the siRNAremaining in the plasma after 168 hours. All the DAR2 (n=2) conjugateshad much faster clearance of the siRNA from the plasma relative to theDAR1 conjugates. As illustrated in FIG. 69B, the DAR1 (n=1) test article(group 9) had a comparable EGFR-Ab plasma clearance profile to the twocontrols (groups 1 and 5). All the DAR2 (n=2) conjugates had much fasterclearance of the antibody from the plasma relative to the DAR1conjugates.

In the above Examples, it was demonstrated the use of lysine andcysteine conjugation strategies to attach the siRNA to the antibody. Inthis example, it was demonstrated the utility of a site specificconjugation strategy and demonstrate the conjugate has comparable PKproperties to non-specific conjugation strategies.

Example 28: 2016-PK-180-HCC827

siRNA Design and Synthesis

EFGR: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against human EGFR. The sequence ofthe guide/antisense strand was complementary to the gene sequencestarting a base position 333 for the human mRNA transcript for EGFR(ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base, sugar and phosphatemodifications were used to optimize the potency of the duplex and reduceimmunogenicity. All siRNA single strands were fully assembled on solidphase using standard phospharamidite chemistry and purified over HPLC.Purified single strands were duplexed to get the double stranded siRNA.The passenger strand contained two conjugation handles, a C6-NH2 at the5′ end and a C6-SH at the 3′ end. Both conjugation handles wereconnected to siRNA passenger strand via phosphorothioate-invertedabasic-phosphorothioate linker, see Example 9 for the chemicalstructure.

Negative control siRNA sequence (scramble): A published (Burke et al.(2014) Pharm. Res., 31(12):3445-60) 21mer duplex with 19 bases ofcomplementarity and 3′ dinucleotide overhangs was used. The sequence (5′to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ IDNO: 2116). Base, sugar and phosphate modifications were used to reduceimmunogenicity and were comparable to those used in the active siRNA.All siRNA single strands were fully assembled on solid phase usingstandard phospharamidite chemistry and purified over HPLC. Purifiedsingle strands were duplexed to get the double stranded siRNA. Thepassenger strand contained two conjugation handles, a C6-NH2 at the 5′end and a C6-SH at the 3′ end. Both conjugation handles were connectedto siRNA passenger strand via a phosphodiester-invertedabasic-phosphodiester linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASCarchitecture-4, as described in Example 9.

In Vivo Study Design

Groups (n=5) of female NCr nu/nu mice bearing subcutaneously (SC) flankHCC827 tumors 100-300 mm³ in volume were treated with one intravenous(i.v.) tail vein injection of siRNA conjugate, while control group (n=5)of the same mice received one i.v. injection of PBS as a vehiclecontrol. Table 40 describes the study design. Mice were sacrificed byCO₂ asphyxiation at 96 hours post-dose. 50 mg pieces of tumor and liver,were collected and snap-frozen in liquid nitrogen. mRNA knockdown intarget tissue was determined using a comparative qPCR assay as describedin Example 2. Total RNA was extracted from the tissue, reversetranscribed and mRNA levels were quantified using TaqMan qPCR, using theappropriately designed primers and probes. PPIB (housekeeping gene) wasused as an internal RNA loading control, results were calculated by thecomparative Ct method, where the difference between the target gene Ctvalue and the PPIB Ct value (ΔCt) is calculated and then furthernormalized relative to the PBS control group by taking a seconddifference (ΔΔCt). Quantitation of plasma and tissue siRNAconcentrations were determined using a stem-loop qPCR assay as describedin Example 2. The antisense strand of the siRNA was reverse transcribedusing a TaqMan MicroRNA reverse transcription kit using asequence-specific stem-loop RT primer. The cDNA from the RT step wasthen utilized for real-time PCR and Ct values were transformed intoplasma and tissue concentrations using the linear equations derived fromthe standard curves.

TABLE 40 siRNA Dose Harvest Dose Volume # of Time Gr Test Article N(mg/kg) ROA (mL/kg) Doses (h) 1 EGFR-Ab(Cys)-EGFR-PEG5k (n = 1) 5 1 IV5.0 1 96 2 EGFR-Ab(Cys)-EGFR-PEG5k (n = 1) 5 0.5 IV 5.0 1 96 3EGFR-Ab(Cys)-EGFR-PEG5k (n = 1) 5 0.25 IV 5.0 1 96 4EGFR-Ab(Cys)-ECL-EGFR-PEG5k (n = 1) 5 1 IV 5.0 1 96 5EGFR-Ab(Cys)-ECL-EGFR-PEG5k (n = 1) 5 0.5 IV 5.0 1 96 6EGFR-Ab(Cys)-ECL-EGFR-PEG5k (n = 1) 5 0.25 IV 5.0 1 96 7EGFR-Ab(Cys)-EGFR-SS-PEG5k (n = 1) 5 1 IV 5.0 1 96 8EGFR-Ab(Cys)-EGFR-SS-PEG5k (n = 1) 5 0.5 IV 5.0 1 96 9EGFR-Ab(Cys)-EGFR-SS-PEG5k (n = 1) 5 0.25 IV 5.0 1 96 10EGFR-Ab(Cys)-ECL-EGFR-SS-PEG5k 5 1 IV 5.0 1 96 (n = 1) 11EGFR-Ab(Cys)-ECL-EGFR-SS-PEG5k 5 0.5 IV 5.0 1 96 (n = 1) 12EGFR-Ab(Cys)-ECL-EGFR-SS-PEG5k 5 0.25 IV 5.0 1 96 (n = 1) 15EGFR-Ab(Cys)-scramble-PEG5k (n = 1) 5 1 IV 5.0 1 96 16 PBS Control 5 —IV 5.0 1 96 Total # of Animals: 80 nu/nu mice with HCC827 tumors

In this in vivo PK study, replacing the SMCC linker between the antibodyand siRNA with an enzymatically cleavable linker and the introduction ofa cleavable disulfide linker between the siRNA and PEG, or thecombination of both were tested. As illustrated in FIG. 70A, all thelinker combination were capable of EGFR mRNA knockdown in the HCC827tumor cells relative to the scrambled control. As illustrated in FIG.70B, all the linker combinations produced comparable siRNA tissueaccumulation in the tumor and liver. As illustrated in FIG. 70C, all theconjugates were capable of maintaining high levels of siRNA in theplasma, with approximately 10% remaining in the plasma after 168 hours.

In this AXBYC example, it was demonstrated that different linkercombinations (“X” and/or “Y”) can be used to conjugate the siRNA to theantibody and PEG.

Example 29: 2016-PK-162-LNCaP

EFGR: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against human EGFR. The sequence ofthe guide/antisense strand was complementary to the gene sequencestarting a base position 333 for the human mRNA transcript for EGFR(ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base, sugar and phosphatemodifications were used to optimize the potency of the duplex and reduceimmunogenicity. All siRNA single strands were fully assembled on solidphase using standard phospharamidite chemistry and purified over HPLC.Purified single strands were duplexed to get the double stranded siRNA.The passenger strand contained two conjugation handles, a C6-NH₂ at the5′ end and a C6-SH at the 3′ end. Both conjugation handles wereconnected to siRNA passenger strand via phosphorothioate-invertedabasic-phosphorothioate linker, see Example 9 for the chemicalstructure.

Negative control siRNA sequence (scramble): A published (Burke et al.(2014) Pharm. Res., 31(12):3445-60) 21mer duplex with 19 bases ofcomplementarity and 3′ dinucleotide overhangs was used. The sequence (5′to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ IDNO: 2116). Base, sugar and phosphate modifications were used to reduceimmunogenicity and were comparable to those used in the active siRNA.All siRNA single strands were fully assembled on solid phase usingstandard phospharamidite chemistry and purified over HPLC. Purifiedsingle strands were duplexed to get the double stranded siRNA. Thepassenger strand contained two conjugation handles, a C6-NH₂ at the 5′end and a C6-SH at the 3′ end. Both conjugation handles were connectedto siRNA passenger strand via a phosphodiester-invertedabasic-phosphodiester linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASCarchitecture-4, as described in Example 9.

In Vivo Study Design

Groups 1-7 (n=5) of female SCID SHO mice bearing subcutaneous flankLNCaP tumors 100-350 mm³ in volume were treated with one intravenous(i.v.) tail vein injection of siRNA conjugate, while control group 8(n=5) of the same mice received one i.v. injection of PBS as a vehiclecontrol. The table below describes the study design. Mice weresacrificed by CO₂ asphyxiation at 96 hours post-dose. 50 mg pieces oftumor and liver, were collected and snap-frozen in liquid nitrogen. mRNAknockdown in target tissue was determined using a comparative qPCR assayas described in Example 2. Total RNA was extracted from the tissue,reverse transcribed and mRNA levels were quantified using TaqMan qPCR,using the appropriately designed primers and probes. PPIB (housekeepinggene) was used as an internal RNA loading control, results werecalculated by the comparative Ct method, where the difference betweenthe target gene Ct value and the PPIB Ct value (ΔCt) is calculated andthen further normalized relative to the PBS control group by taking asecond difference (ΔΔCt). Quantitation of tissue siRNA concentrationswere determined using a stem-loop qPCR assay as described in Example 2.The antisense strand of the siRNA was reverse transcribed using a TaqManMicroRNA reverse transcription kit using a sequence-specific stem-loopRT primer. The cDNA from the RT step was then utilized for real-time PCRand Ct values were transformed into plasma or tissue concentrationsusing the linear equations derived from the standard curves.

TABLE 41 siRNA Dose Harvest Dose Volume # of Time Group Test Article N(mg/kg) ROA (mL/kg) Doses (h) 1 PSMA-Ab(Lys)-SS-EGFR-PEG5k 5 1 IV 5.0 196 (n = 1) 2 PSMA-Ab(Lys)-SS-EGFR-PEG5k 5 0.5 IV 5.0 1 96 (n = 1) 3PSMA-Ab(Cys)-ECL-EGFR-PEG5k 5 1 IV 5.0 1 96 (n = 1) 4PSMA-Ab(Cys)-ECL-EGFR-PEG5k 5 0.5 IV 5.0 1 96 (n = 1) 5PSMA-Ab(Cys)-EGFR-PEG5k 5 1 IV 5.0 1 96 (n = 1) FRESH 6PSMA-Ab(Cys)-EGFR-PEG5k 5 1 IV 5.0 1 96 (n = 1) FROZEN 7PSMA-Ab(Cys)-svcramble-PEG5k 5 1 IV 5.0 1 96 (n = 1) 8 PBS Control 5 —IV 5.0 1 96 Total # of Animals: 40 SCID SHO mice with LNCaP tumors

In this in vivo PK study, a disulfide (SS), enzymatically cleavable(ECL) or SMCC linker was used between the antibody and siRNA. Asillustrated in graph 1 on slide 42, the tumor tissue accumulation of thesiRNA was reduced when the cleavable disulfide leaker was used insteadof the ECL or SMCC linkers. As illustrated on graph 2 on slide 42, theECL linker strategy produced EGFR mRNA knockdown in the LNCaP tumorcells relative to the scrambled control. However, the SS linker failedto produce EGFR mRNA knockdown in the LNCaP tumor cells relative to thescrambled control. In addition to these linker experiments, thefeasibility of −80° C. storage of the ASC was examined. The Formulationwas snap-frozen in liquid nitrogen at 5 mg/ml antibody concentration,thawed at room temperature after 30 days storage at −80° C. and dilutedto the required dosing concentration prior to administration. Asillustrated on graph 3 on slide 42, the construct stored at −80° C.,thawed prior to administration, retained its ability to produce EGFRmRNA knockdown in the LNCaP tumor cells relative to the scrambledcontrol.

In this AXBYC example, it was demonstrated that an ECL linker (“X”) canbe used to conjugate the antibody to the siRNA and that an ASC can bestored at −80° C. for 1 month and thawed prior to administration.

Example 30: 2016-PK-181-HCC827

siRNA Design and Synthesis

EFGR: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against human EGFR. The sequence ofthe guide/antisense strand was complementary to the gene sequencestarting a base position 333 for the human mRNA transcript for EGFR(ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base, sugar and phosphatemodifications that are well described in the field of RNAi were used tooptimize the potency of the duplex and reduce immunogenicity. All siRNAsingle strands were fully assembled on solid phase using standardphospharamidite chemistry and purified over HPLC. Purified singlestrands were duplexed to get the double stranded siRNA. The passengerstrand contained two conjugation handles, a C6-NH₂ at the 5′ end and aC6-SH at the 3′ end. Both conjugation handles were connected to siRNApassenger strand via phosphorothioate-inverted abasic-phosphorothioatelinker, see Example 9 for the chemical structure.

Negative control siRNA sequence (scramble): A published (Burke et al.(2014) Pharm. Res., 31(12):3445-60) 21 mer duplex with 19 bases ofcomplementarity and 3′ dinucleotide overhangs was used. The sequence (5′to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ IDNO: 2116). Base, sugar and phosphate modifications were used to reduceimmunogenicity and were comparable to those used in the active siRNA.All siRNA single strands were fully assembled on solid phase usingstandard phospharamidite chemistry and purified over HPLC. Purifiedsingle strands were duplexed to get the double stranded siRNA. Thepassenger strand contained two conjugation handles, a C6-NH₂ at the 5′end and a C6-SH at the 3′ end. Both conjugation handles were connectedto siRNA passenger strand via a phosphodiester-invertedabasic-phosphodiester linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASCarchitecture-4, as described in Example 9.

In Vivo Study Design

Groups (n=5) of female NCr nu/nu mice bearing subcutaneously (SC) flankHCC827 tumors 100-300 mm³ in volume were treated with one intravenous(i.v.) tail vein injection of siRNA conjugate, while control group (n=5)of the same mice received one i.v. injection of PBS as a vehiclecontrol. Table 42 describes the study design. Mice were sacrificed byCO₂ asphyxiation at 96 hours post-dose. 50 mg pieces of tumor and liver,were collected and snap-frozen in liquid nitrogen. mRNA knockdown intarget tissue was determined using a comparative qPCR assay as describedin Example 2. Total RNA was extracted from the tissue, reversetranscribed and mRNA levels were quantified using TaqMan qPCR, using theappropriately designed primers and probes. PPIB (housekeeping gene) wasused as an internal RNA loading control, results were calculated by thecomparative Ct method, where the difference between the target gene Ctvalue and the PPIB Ct value (ΔCt) is calculated and then furthernormalized relative to the PBS control group by taking a seconddifference (ΔΔCt). Quantitation of tissue siRNA concentrations weredetermined using a stem-loop qPCR assay as described in the methodssection. The antisense strand of the siRNA was reverse transcribed usinga TaqMan MicroRNA reverse transcription kit using a sequence-specificstem-loop RT primer. The cDNA from the RT step was then utilized forreal-time PCR and Ct values were transformed into tissue concentrationsusing the linear equations derived from the standard curves.

TABLE 42 siRNA Dose Harvest Dose Volume # of Time Group Test Article N(mg/kg) ROA (mL/kg) Doses (h) 1 EGFR-Ab(Cys)-EGFR-PEG5k (n = 1) 5 1 IV5.0 1 96 2 EGFR-Ab(Cys)-EGFR-PEG5k (n = 1) 5 0.5 IV 5.0 1 96 3EGFR-Ab(Cys)-SS-EGFR-PEG5k (n = 1) 5 1 IV 5.0 1 96 4EGFR-Ab(Cys)-SS-EGFR-PEG5k (n = 1) 5 0.5 IV 5.0 1 96 6EGFR-Ab(Cys)-scramble-PEG5k (n = 1) 5 1 IV 5.0 1 96 7EGFR-Ab(Cys)-scramble-PEG5k (n = 1) 5 0.5 IV 5.0 1 96 8 PBS Control 5 —IV 5.0 1 96 Total # of Animals: 80 nu/nu mice with HCC827 tumors

In this in vivo PK study, a disulfide or SMCC linker was used betweenthe antibody and siRNA. As illustrated in FIG. 72A, the tumor tissueaccumulation of the siRNA was reduced when the cleavable disulfideleaker was used instead of the more stable SMCC linker. As illustratedin FIG. 72B, both linker strategies were capable of producing EGFR mRNAknockdown in the HCC827 tumor cells relative to the scrambled control.

In this AXBYC example, it was demonstrated the use of a cleavabledisulfide linker (“X”) between the antibody and siRNA.

Example 31: 2016-PK-220-WT

siRNA Design and Synthesis

KRAS: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against human KRAS. The sequence ofthe guide/antisense strand was complementary to the gene sequencestarting a base position 237 for the human mRNA transcript for KRAS(Guide strand sequence: UGAAUUAGCUGUAUCGUCAUU; SEQ ID NO: 2088). Base,sugar and phosphate modifications were used to optimize the potency ofthe duplex and reduce immunogenicity. All siRNA single strands werefully assembled on solid phase using standard phospharamidite chemistryand purified over HPLC. Purified single strands were duplexed to get thedouble stranded siRNA. The passenger strand contained two conjugationhandles, a C6-NH₂ at the 5′ end and a C6-SH at the 3′ end. Bothconjugation handles were connected to siRNA passenger strand viaphosphorothioate-inverted abasic-phosphorothioate linker, see Example 9for the chemical structure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASCarchitecture-4, as described in Example 9.

In Vivo Study Design

Groups (n=4) of wild-type female CD-1 mice were treated with oneintravenous (i.v.) tail vein injections of siRNA conjugates. Treatmentgroups received 0.5 mg/kg (based on the weight of siRNA) and all groupswere administered a dose volume of 5.0 mL/kg. Table 43 illustrates thestudy design in more detail. Non-terminal blood samples were collectedat 5, 30, and 180 minutes post-dose via puncture of the retro-orbitalplexus and centrifuged to generate plasma for PK analysis. Mice weresacrificed by CO₂ asphyxiation at 24, 96, or 168 hours post-dose.Terminal blood samples were collected via cardiac puncture and processedto generate plasma for PK analysis. Quantitation of plasma siRNAconcentrations were determined using a stem-loop qPCR assay as describedin Example 2. The antisense strand of the siRNA was reverse transcribedusing a TaqMan MicroRNA reverse transcription kit using asequence-specific stem-loop RT primer. The cDNA from the RT step wasthen utilized for real-time PCR and Ct values were transformed intoplasma or tissue concentrations using the linear equations derived fromthe standard curves. Plasma concentrations of antibody were determinedusing an ELISA assay.

TABLE 43 siRNA Dose Survival Terminal Dose Volume # of Bleed Bleed GroupTest Article N (mg/kg) ROA (mL/kg) Doses (min) (h) 1 EGFR-Ab(Lys)-SPDP-4 0.5 IV 5.0 1 5 24 2 KRAS-PEG5k (n = 1) 4 0.5 IV 5.0 1 30 96 3 4 0.5 IV5.0 1 180 168 4 EGFR-Ab(Cys)-SPDP- 4 0.5 IV 5.0 1 5 24 5 KRAS-PEG5k (n= 1) 4 0.5 IV 5.0 1 30 96 6 4 0.5 IV 5.0 1 180 168 7 EGFR-Ab(Cys)-SMPT-4 0.5 IV 5.0 1 5 24 8 KRAS-PEG5k (n = 1) 4 0.5 IV 5.0 1 30 96 9 4 0.5 IV5.0 1 180 168 10 EGFR-Ab(Cys)-SS(methyl)- 4 0.5 IV 5.0 1 5 24 11KRAS-PEG5k (n = 1) 4 0.5 IV 5.0 1 30 96 12 4 0.5 IV 5.0 1 180 168 13EGFR-Ab(Cys)- 4 0.5 IV 5.0 1 5 24 14 SS(dimethyl)-KRAS-PEG5k 4 0.5 IV5.0 1 30 96 15 (n = 1) 4 0.5 IV 5.0 1 180 168 Total # of Animals: 60 WTmice CD-1

In this in vivo PK study, different disulfide linkers were explored,with varying degrees of steric hindrance, to understand how the rate ofdisulfide cleavage impacts ASC plasma PK. As illustrated in FIG. 73A,the clearance of the siRNA from the plasma was modulated by varying thedegree of steric hindrance of the disulfide linker. FIG. 73B illustratesthe clearance of the antibody zalutumumab from the plasma.

In this example, it was demonstrated biological activity with a range ofdifferent AXBYC conjugates in which a range of different disulfidelinkers (“X”) can be used to conjugate the siRNA to the antibody.

Example 32: 2016-PK-256-WT

siRNA Design and Synthesis

KRAS: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against human KRAS. The sequence ofthe guide/antisense strand was complementary to the gene sequencestarting a base position 237 for the human mRNA transcript for KRAS(Guide strand sequence: UGAAUUAGCUGUAUCGUCAUU; SEQ ID NO: 2088). Base,sugar and phosphate modifications were used to optimize the potency ofthe duplex and reduce immunogenicity. All siRNA single strands werefully assembled on solid phase using standard phospharamidite chemistryand purified over HPLC. Purified single strands were duplexed to get thedouble stranded siRNA. The passenger strand contained two conjugationhandles, a C6-NH2 at the 5′ end and a C6-SH at the 3′ end. Bothconjugation handles were connected to siRNA passenger strand viaphosphorothioate-inverted abasic-phosphorothioate linker, see Example 9for the chemical structure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASCarchitecture-4, as described in Example 9.

In Vivo Study Design

Groups (n=4) of wild-type female CD-1 mice were treated with oneintravenous (i.v.) tail vein injections of siRNA conjugates. Treatmentgroups received 0.5 mg/kg (based on the weight of siRNA) and all groupswere administered a dose volume of 5.0 mL/kg. Table 44 illustrates thestudy design in more detail. Non-terminal blood samples were collectedat 0.25, 3, and 24 hours post-dose via puncture of the retro-orbitalplexus and centrifuged to generate plasma for PK analysis. Mice weresacrificed by CO₂ asphyxiation at 72, 96, or 168 h post-dose. Terminalblood samples were collected via cardiac puncture and processed togenerate plasma for PK analysis. Quantitation of plasma siRNAconcentrations were determined using a stem-loop qPCR assay as describedin Example 2. The antisense strand of the siRNA was reverse transcribedusing a TaqMan MicroRNA reverse transcription kit using asequence-specific stem-loop RT primer. The cDNA from the RT step wasthen utilized for real-time PCR and Ct values were transformed intoplasma or tissue concentrations using the linear equations derived fromthe standard curves. Plasma concentrations of antibody were determinedusing an ELISA assay.

TABLE 44 siRNA Dose Survival Terminal Dose Volume # of Bleed Bleed GroupTest Article N (mg/kg) ROA (mL/kg) Doses (h) (h) 1 EGFR-Ab(Cys)-SMCC- 40.5 IV 5.0 1 0.25 72 KRAS-PEG5k (n = 1) 2 EGFR-Ab(Cys)-SMCC- 4 0.5 IV5.0 1 3 96 KRAS-PEG5k (n = 1) 3 EGFR-Ab(Cys)-SMCC- 4 0.5 IV 5.0 1 24 168KRAS-PEG5k (n = 1) 4 EGFR-Ab(Cys)-CBTF- 4 0.5 IV 5.0 1 0.25 72KRAS-PEG5k (n = 1) 5 EGFR-Ab(Cys)-CBTF- 4 0.5 IV 5.0 1 3 96 KRAS-PEG5k(n = 1) 6 EGFR-Ab(Cys)-CBTF- 4 0.5 IV 5.0 1 24 168 KRAS-PEG5k (n = 1) 7EGFR-Ab(Cys)-MBS- 4 0.5 IV 5.0 1 0.25 72 KRAS-PEG5k (n = 1) 8EGFR-Ab(Cys)-MBS- 4 0.5 IV 5.0 1 3 96 KRAS-PEG5k (n = 1) 9EGFR-Ab(Cys)-MBS- 4 0.5 IV 5.0 1 24 168 KRAS-PEG5k (n = 1) Total # ofAnimals: 60 WT mice CD-1

In this in vivo PK study a range of different linkers between theantibody and siRNA were tested to determine the effect on plasmaclearance. As illustrated on the graph on slide 45, all the conjugateswere capable of maintaining high levels of siRNA in the plasma, withgreater than 10% remaining in the plasma after 168 hours.

In this example, it was demonstrated biological activity with a range ofdifferent AXBYC conjugates in which a range of different linkers (“Y”)can be used to conjugate the siRNA to the antibody while maintaining theimproved plasma kinetics over those historically observed forunconjugated siRNA.

Example 33: 2016-PK-237-HCC827

siRNA Design and Synthesis

EFGR: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against human EGFR. The sequence ofthe guide/antisense strand was complementary to the gene sequencestarting a base position 333 for the human mRNA transcript for EGFR(Guide strad sequence: ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base,sugar and phosphate modifications were used to optimize the potency ofthe duplex and reduce immunogenicity. All siRNA single strands werefully assembled on solid phase using standard phospharamidite chemistryand purified over HPLC. Purified single strands were duplexed to get thedouble stranded siRNA.

Two different passenger strands were made containing two conjugationhandles (C6-NH₂ and C6-SH) in two different orientations (S5′-EGFR-3′Nand N5′-EGFR-3′S). In the N5′-EGFR-3′S passenger strand both conjugationhandles were connected to siRNA passenger strand viaphosphorothioate-inverted abasic-phosphorothioate linker, see Example 9for the chemical structure. In the S5′-EGFR-3′N passenger strand bothconjugation handles were connected to siRNA passenger strand viaphosphodiester-inverted abasic-phosphorothioate linker. The C6-NH₂ andC6-SH were connected through the phosphodiester, see Example 9 for thechemical structure.

ASC Synthesis and Characterization

The conjugate for groups 1-3 was made and purified as a DAR1 (n=1) usingASC architecture-4, as described in Example 9. The conjugate for groups4-6 was made and purified as a DAR1 (n=1) using ASC architecture-2, asdescribed in Example 9.

In Vivo Study Design

Groups (n=5) of female NCr nu/nu mice bearing subcutaneously (SC) flankHCC827 tumors 100-300 mm³ in volume were treated with one intravenous(i.v.) tail vein injection of siRNA conjugate, while control group (n=5)of the same mice received one i.v. injection of PBS as a vehiclecontrol. Table 45 describes the study design. Mice were sacrificed byCO₂ asphyxiation at 72, 96, and 168 hours post-dose. 50 mg pieces oftumor and liver, were collected and snap-frozen in liquid nitrogen. mRNAknockdown in target tissue was determined using a comparative qPCR assayas described in Example 2. Total RNA was extracted from the tissue,reverse transcribed and mRNA levels were quantified using TaqMan qPCR,using the appropriately designed primers and probes. PPIB (housekeepinggene) was used as an internal RNA loading control, results werecalculated by the comparative Ct method, where the difference betweenthe target gene Ct value and the PPIB Ct value (ΔCt) is calculated andthen further normalized relative to the PBS control group by taking asecond difference (ΔΔCt). Quantitation of tissue and plasma siRNAconcentrations were determined using a stem-loop qPCR assay as describedin Example 2. The antisense strand of the siRNA was reverse transcribedusing a TaqMan MicroRNA reverse transcription kit using asequence-specific stem-loop RT primer. The cDNA from the RT step wasthen utilized for real-time PCR and Ct values were transformed intoplasma or tissue concentrations using the linear equations derived fromthe standard curves.

TABLE 45 siRNA Dose Survival Terminal Harvest Dose Volume # of BleedBleed Time Gr Test Article N (mg/kg) ROA (mL/kg) Doses (min) (h) (h) 1EGFR-Ab(Cys)-S5′- 5 0.5 IV 5.0 1 0.25 72 72 EGFR-3′N-PEG5k (n = 1) 2EGFR-Ab(Cys)-S5′- 5 0.5 IV 5.0 1 3 96 96 EGFR-3′N-PEG5k (n = 1) 3EGFR-Ab(Cys)-S5′- 5 0.5 IV 5.0 1 24 168 168 EGFR-3′N-PEG5k (n = 1) 4EGFR-Ab(Cys)-N5′- 5 0.5 IV 5.0 1 0.25 72 72 EGFR-3′S-PEG5k (n = 1) 5EGFR-Ab(Cys)-N5′- 5 0.5 IV 5.0 1 3 96 96 EGFR-3′S-PEG5k (n = 1) 6EGFR-Ab(Cys)-N5′- 5 0.5 IV 5.0 1 24 168 168 EGFR-3′S-PEG5k (n = 1) 7 PBSControl 5 — IV 5.0 1 — — 96 Total # of Animals: 65 nu/nu mice withHCC827 tumors

In this in vivo PK study the biological outcome of changes in theorientation of the conjugation site of the antibody and PEG (5′ or 3′)onto the siRNA were evaluated. In addition, the biological outcome ofusing a lysine or cysteine to attach the linker to the antibody wasevaluated As illustrated FIG. 75A, both orientations of siRNA producedcomparable EGFR tumor knockdown. As illustrated FIG. 75B and FIG. 75C,both orientations produced comparable siRNA tissue accumulation in thetumor and liver. As illustrated in FIG. 75D, both orientations produce acomparable plasma clearance kinetics.

As highlighted in FIG. 54, it was demonstrated biological activity withthe A-X—B—Y—C conjugate with a range of different antibodies and siRNAcargos that are capable of in vivo biological activity in a range ofdifferent tissue targets. In this example, it was demonstrated that theantibody can be conjugated onto the 5′ and 3′ ends of the passengerstrand of the siRNA and while maintaining the biological activity of theEGFR siRNA and tissue distribution.

Example 34: 2016-PK-259-WT

siRNA Design and Synthesis

HPRT: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against human HPRT. The sequence ofthe guide/antisense strand was complementary to the gene sequencestarting a base position 425 for the human mRNA transcript for HPRT(guide strand sequence: UUAAAAUCUACAGUCAUAGUU; SEQ ID NO: 2104). Base,sugar and phosphate modifications were used to optimize the potency ofthe duplex and reduce immunogenicity. All siRNA single strands werefully assembled on solid phase using standard phospharamidite chemistryand purified over HPLC. Purified single strands were duplexed to get thedouble stranded siRNA. Two different passenger strands were madecontaining two conjugation handles (C6-NH₂ and C6-SH) in two differentorientations (S5′-HPRT-3′N and N5′-HPRT-3′S). Both conjugation handleswere connected to siRNA passenger strand via phosphodiester-invertedabasic-phosphorothioate linker. The C6-NH₂ and C6-SH were connectedthrough the phosphodiester, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

The conjugate for groups 1-3 was made and purified as a DAR1 (n=1) usingASC architecture-4, as described in Example 9. The conjugate for groups4-6 was made and purified as a DAR1 (n=1) using ASC architecture-2, asdescribed in Example 9. The conjugate for groups 7-9 was made andpurified as a DAR1 (n=1) using ASC architecture-1, as described inExample 9. The conjugate for groups 10-12 was made and purified as aDAR1 (n=1) using ASC architecture-3, as described in Example 9.

In Vivo Study Design

Groups (n=4) of wild-type female CD-1 mice were treated with oneintravenous (i.v.) tail vein injections of siRNA conjugates, while thecontrol group (n=5) of the same mice received one i.v. injection of PBSas a vehicle control. Table 46 illustrates the study design in moredetail. 50 mg pieces of tissue, were collected and snap-frozen in liquidnitrogen. mRNA knockdown in target tissue was determined using acomparative qPCR assay as described in Example 2. Total RNA wasextracted from the tissue, reverse transcribed and mRNA levels werequantified using TaqMan qPCR, using the appropriately designed primersand probes. PPIB (housekeeping gene) was used as an internal RNA loadingcontrol, results were calculated by the comparative Ct method, where thedifference between the target gene Ct value and the PPIB Ct value (ΔCt)is calculated and then further normalized relative to the PBS controlgroup by taking a second difference (ΔΔCt). Quantitation of tissue siRNAconcentrations were determined using a stem-loop qPCR assay as describedin Example 2. The antisense strand of the siRNA was reverse transcribedusing a TaqMan MicroRNA reverse transcription kit using asequence-specific stem-loop RT primer. The cDNA from the RT step wasthen utilized for real-time PCR and Ct values were transformed intoplasma or tissue concentrations using the linear equations derived fromthe standard curves.

TABLE 46 siRNA Dose Harvest Dose Volume # of Time Group Test Article N(mg/kg) ROA (mL/kg) Doses (h) 1 Anti-B cell Ab(Cys)-N5′-HPRT-3′S- 4 3 IV5.0 1 96 PEG5k (n = 1) 2 Anti-B cell Ab(Cys)-N5′-HPRT-3′S- 4 1 IV 5.0 196 PEG5k (n = 1) 3 Anti-B cell Ab(Cys)-N5′-HPRT-3′S- 4 0.3 IV 5.0 1 96PEG5k (n = 1) 4 Anti-B cell Ab(Cys)-N3′-HPRT-5′S- 4 3 IV 5.0 1 96 PEG5k(n = 1) 5 Anti-B cell Ab(Cys)-N3′-HPRT-5′S- 4 1 IV 5.0 1 96 PEG5k (n= 1) 6 Anti-B cell Ab(Cys)-N3′-HPRT-5′S- 4 0.3 IV 5.0 1 96 PEG5k (n = 1)7 Anti-B cell Ab(Lys)-S3′-HPRT-5′N- 4 2 IV 5.0 1 96 PEG5k (n = 1) 8Anti-B cell Ab(Lys)-S3′-HPRT-5′N- 4 0.75 IV 5.0 1 96 PEG5k (n = 1) 9Anti-B cell Ab(Lys)-S3′-HPRT-5′N- 4 0.25 IV 5.0 1 96 PEG5k (n = 1) 10Anti-B cell Ab(Lys)-S5′-HPRT-3′N- 4 2 IV 5.0 1 96 PEG5k (n = 1) 11Anti-B cell Ab(Lys)-S5′-HPRT-3′N- 4 0.75 IV 5.0 1 96 PEG5k (n = 1) 12Anti-B cell Ab(Lys)-S5′-HPRT-3′N- 4 0.25 IV 5.0 1 96 PEG5k (n = 1) 13PBS Control 5 — IV 5.0 1 96 Total # of Animals: 53 WT mice (CD-1)

In the in vivo PK study the biological outcome of changes in theorientation of the conjugation site of the antibody and PEG (5′ or 3′)onto the siRNA were evaluated. In addition, the biological outcome ofusing a lysine or cysteine to attach the linker to the antibody wasevaluated. As illustrated in FIG. 76A-FIG. 76D, all the combinations ofmaking the antibody conjugates produced comparable HPRT knockdown in thefour tissue compartments measured. As illustrated in FIG. 77A-FIG. 77D,all the combinations of making the antibody conjugates producedcomparable siRNA tissue accumulation in the different compartmentsmeasured.

In this example, it was demonstrated that a variety of differentconjugation strategies to the siRNA and antibody can be used in theA-X—B—Y—C format while maintaining the biological activity of the HPRTsiRNA and tissue distribution.

Example 35: 2016-PK-267-WT

siRNA Design and Synthesis

CTNNB1: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against human CTNNB1. The sequenceof the guide/antisense strand was complementary to the gene sequencestarting a base position 1797 for the human mRNA transcript for CTNNB1(guide strand sequence: UUUCGAAUCAAUCCAACAGUU; SEQ ID NO: 2098). Base,sugar and phosphate modifications were used to optimize the potency ofthe duplex and reduce immunogenicity. All siRNA single strands werefully assembled on solid phase using standard phospharamidite chemistryand purified over HPLC. Purified single strands were duplexed to get thedouble stranded siRNA.

Two different passenger strands were made containing two conjugationhandles (C6-NH₂ and C6-SH) in two different orientations (S5′-CTNNB1-3′Nand N5′-CTNNB1-3′S). Both conjugation handles were connected to siRNApassenger strand via phosphodiester-inverted abasic-phosphorothioatelinker. The C6-NH₂ and C6-SH were connected through the phosphodiester,see Example 9 for the chemical structure.

ASC Synthesis and Characterization

The conjugate for groups 1-3 was made and purified as a DAR1 (n=1) usingASC architecture-4, as described in Example 9. The conjugate for groups4-6 was made and purified as a DAR1 (n=1) using ASC architecture-3, asdescribed in Example 9. The conjugate for groups 7-9 was made andpurified as a DAR1 (n=1) using ASC architecture-2, as described inExample 9. The conjugate for groups 10-12 was made and purified as aDAR1 (n=1) using ASC architecture-1, as described in Example 9.

In Vivo Study Design

Groups (n=4) of wild-type female CD-1 mice were treated with oneintravenous (i.v.) tail vein injections of siRNA conjugates, while thecontrol group (n=5) of the same mice received one i.v. injection of PBSas a vehicle control. Table 47 illustrates the study design in moredetail. 50 mg pieces of tissue, were collected and snap-frozen in liquidnitrogen. mRNA knockdown in target tissue was determined using acomparative qPCR assay as described in Example 2. Total RNA wasextracted from the tissue, reverse transcribed and mRNA levels werequantified using TaqMan qPCR, using the appropriately designed primersand probes. PPIB (housekeeping gene) was used as an internal RNA loadingcontrol, results were calculated by the comparative Ct method, where thedifference between the target gene Ct value and the PPIB Ct value (ΔCt)is calculated and then further normalized relative to the PBS controlgroup by taking a second difference (ΔΔCt).

TABLE 47 siRNA Dose Harvest Dose Volume # of Time Group Test Article N(mg/kg) ROA (mL/kg) Doses (h) 1 Anti-B cell Ab(Cys)-N5′- 4 3 IV 5.0 1 96CTNNB1-3′S-PEG5k (n = 1) 2 Anti-B cell Ab(Cys)-N5′- 4 1 IV 5.0 1 96CTNNB1-3′S-PEG5k (n = 1) 3 Anti-B cell Ab(Cys)-N5′- 4 0.3 IV 5.0 1 96CTNNB1-3′S-PEG5k (n = 1) 4 Anti-B cell Ab(Lys)-S5′- 4 3 IV 5.0 1 96CTNNB1-3′N-PEG5k (n = 1) 5 Anti-B cell Ab(Lys)-S5′- 4 1 IV 5.0 1 96CTNNB1-3′N-PEG5k (n = 1) 6 Anti-B cell Ab(Lys)-S5′- 4 0.3 IV 5.0 1 96CTNNB1-3′N-PEG5k (n = 1) 7 Anti-B cell Ab(Cys)-N3′- 4 3 IV 5.0 1 96CTNNB1-5′S-PEG5k (n = 1) 8 Anti-B cell Ab(Cys)-N3′- 4 1 IV 5.0 1 96CTNNB1-5′S-PEG5k (n = 1) 9 Anti-B cell Ab(Cys)-N3′- 4 0.3 IV 5.0 1 96CTNNB1-5′S-PEG5k (n = 1) 10 Anti-B cell Ab(Lys)-S3′- 4 3 IV 5.0 1 96CTNNB1-5′N-PEG5k (n = 1) 11 Anti-B cell Ab(Lys)-S3′- 4 1 IV 5.0 1 96CTNNB1-5′N-PEG5k (n = 1) 12 Anti-B cell Ab(Lys)-S3′- 4 0.3 IV 5.0 1 96CTNNB1-5′N-PEG5k (n = 1) 13 PBS Control 5 — IV 5.0 1 96 Total # ofAnimals: 53 WT mice (CD-1)

In this in vivo PK study, the biological outcome of changes in theorientation of the conjugation site of the antibody and PEG (5′ or 3′)onto the siRNA and the biological outcome of using a lysine or cysteineto attach the linker to the antibody were evaluated. As illustrated inFIG. 78A-FIG. 78D, all the combinations of making the antibodyconjugates produced comparable CTNNB1 knockdown in the four tissuecompartments measured.

In this example, it was demonstrated that a variety of differentconjugation strategies to the siRNA and antibody can be used in theA-X—B—Y—C format while maintaining the biological activity of the CTNNB1siRNA.

Example 36: 2016-PK-188-PK

EFGR: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against human EGFR. The sequence ofthe guide/antisense strand was complementary to the gene sequencestarting a base position 333 for the human mRNA transcript for EGFR(ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base, sugar and phosphatemodifications were used to optimize the potency of the duplex and reduceimmunogenicity. All siRNA single strands were fully assembled on solidphase using standard phospharamidite chemistry and purified over HPLC.Purified single strands were duplexed to get the double stranded siRNA.The passenger strand contained two conjugation handles, a C6-NH₂ at the5′ end and a C6-SH at the 3′ end. Both conjugation handles wereconnected to siRNA passenger strand via phosphorothioate-invertedabasic-phosphorothioate linker, see Example 9 for the chemicalstructure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASCarchitecture-4, as described in Example 9.

In Vivo Study Design

Groups (n=4) of wild-type female CD-1 mice were treated with oneintravenous (i.v.) tail vein injections of siRNA conjugates. Treatmentgroups received 0.5 mg/kg (based on the weight of siRNA) and all groupswere administered a dose volume of 5.0 mL/kg. Table 48 illustrates thestudy design in more detail. Non-terminal blood samples were collectedat 5, 30, and 180 minutes post-dose via puncture of the retro-orbitalplexus and centrifuged to generate plasma for PK analysis. Mice weresacrificed by CO₂ asphyxiation at 24, 96, or 168 h post-dose. Terminalblood samples were collected via cardiac puncture and processed togenerate plasma for PK analysis. Quantitation of plasma siRNAconcentrations were determined using a stem-loop qPCR assay as describedin Example 2. The antisense strand of the siRNA was reverse transcribedusing a TaqMan MicroRNA reverse transcription kit using asequence-specific stem-loop RT primer. The cDNA from the RT step wasthen utilized for real-time PCR and Ct values were transformed intoplasma or tissue concentrations using the linear equations derived fromthe standard curves.

TABLE 48 Dose Survival Terminal Volume # of Bleed Bleed Gr Test ArticleN ROA (mL/kg) Doses (min) (h) 1 EGFR-Ab(Cys)-EGFR-PEG5k (n = 1) 4 IV 5.01 5 24 2 4 IV 5.0 1 30 96 3 4 IV 5.0 1 180 168 4EGFR-Ab(Cys)-ECL-EGFR-PEG5k (n = 1) 4 IV 5.0 1 5 24 5 4 IV 5.0 1 30 96 64 IV 5.0 1 180 168 7 EGFR-Ab(Cys)-EGFR-SS-PEG5k (n = 1) 4 IV 5.0 1 5 248 4 IV 5.0 1 30 96 9 4 IV 5.0 1 180 168 10EGFR-Ab(Cys)-ECL-EGFR-SS-PEG5k 4 IV 5.0 1 5 24 11 (n = 1) 4 IV 5.0 1 3096 12 4 IV 5.0 1 180 168 Total # of Animals: 48 WT mice CD-1

As illustrated in FIG. 79, all the ASC with the different cleavablelinker configurations achieved equivalent plasma PK profiles, withapproximately 10% of the siRNA remaining 168 hours after administration.

In this example, it was demonstrated biological activity with a range ofA-X—B—Y—C conjugates in which a variety of different linker strategies(component X and Y) were used to conjugate the PEG and antibody to thesiRNA passenger strand.

Example 37: 2016-PK-201-LNCaP

siRNA Design and Synthesis

EFGR: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against human EGFR. The sequence ofthe guide/antisense strand was complementary to the gene sequencestarting a base position 333 for the human mRNA transcript for EGFR(ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base, sugar and phosphatemodifications were used to optimize the potency of the duplex and reduceimmunogenicity. All siRNA single strands were fully assembled on solidphase using standard phospharamidite chemistry and purified over HPLC.Purified single strands were duplexed to get the double stranded siRNA.The passenger strand contained two conjugation handles, a C6-NH2 at the5′ end and a C6-SH at the 3′ end. Both conjugation handles wereconnected to siRNA passenger strand via phosphorothioate-invertedabasic-phosphorothioate linker, see Example 9 for the chemicalstructure.

Negative control siRNA sequence (scramble): A published (Burke et al.(2014) Pharm. Res., 31(12):3445-60) 21mer duplex with 19 bases ofcomplementarity and 3′ dinucleotide overhangs was used. The sequence (5′to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ IDNO: 2116). Base, sugar and phosphate modifications were used to reduceimmunogenicity and were comparable to those used in the active siRNA.All siRNA single strands were fully assembled on solid phase usingstandard phospharamidite chemistry and purified over HPLC. Purifiedsingle strands were duplexed to get the double stranded siRNA. Thepassenger strand contained two conjugation handles, a C6-NH₂ at the 5′end and a C6-SH at the 3′ end. Both conjugation handles were connectedto siRNA passenger strand via a phosphodiester-invertedabasic-phosphodiester linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASCarchitecture-4, as described in Example 9.

In Vivo Study Design

Groups 1-7 (n=5) of female SCID SHO mice bearing subcutaneous flankLNCaP tumors 100-350 mm³ in volume were treated with one intravenous(i.v.) tail vein injection of siRNA conjugate, while control group 8(n=5) of the same mice received one i.v. injection of PBS as a vehiclecontrol. Table 49 describes the study design. Mice were sacrificed byCO₂ asphyxiation at 96 hours post-dose. 50 mg pieces of tumor and liver,were collected and snap-frozen in liquid nitrogen. mRNA knockdown intarget tissue was determined using a comparative qPCR assay as describedin Example 2. Total RNA was extracted from the tissue, reversetranscribed and mRNA levels were quantified using TaqMan qPCR, using theappropriately designed primers and probes. PPIB (housekeeping gene) wasused as an internal RNA loading control, results were calculated by thecomparative Ct method, where the difference between the target gene Ctvalue and the PPIB Ct value (ΔCt) is calculated and then furthernormalized relative to the PBS control group by taking a seconddifference (ΔΔCt). Quantitation of tissue siRNA concentrations weredetermined using a stem-loop qPCR assay as described in Example 2. Theantisense strand of the siRNA was reverse transcribed using a TaqManMicroRNA reverse transcription kit using a sequence-specific stem-loopRT primer. The cDNA from the RT step was then utilized for real-time PCRand Ct values were transformed into plasma or tissue concentrationsusing the linear equations derived from the standard curves.

TABLE 49 siRNA Dose Harvest Dose Volume # of Time Group Test Article N(mg/kg) ROA (mL/kg) Doses (h) 1 PSMA-Ab(Cys)- 5 1 IV 5.0 1 96EGFR-SS-PEG5k (n = 1) 2 PSMA-Ab(Cys)- 5 0.5 IV 5.0 1 96 EGFR-SS-PEG5k (n= 1) 3 PSMA-Ab(Cys)- 5 1 IV 5.0 1 96 EGFR-ECL-PEG5k (n = 1) 4PSMA-Ab(Cys)- 5 0.5 IV 5.0 1 96 EGFR-ECL-PEG5k (n = 1) 5 PSAM-Ab(Cys)- 51 IV 5.0 1 96 EGFR-PEG5k (n = 1) 6 PSAM-Ab(Cys)- 5 0.5 IV 5.0 1 96EGFR-PEG5k (n = 1) 7 PSMA-Ab(Cys)- 5 1 IV 5.0 1 96 scramble-PEG5k (n= 1) 8 PBS Control 5 — IV 5.0 1 96 Total # of Animals: 40 SCID SHO micewith LNCaP tumors

As illustrated in FIG. 80A, a variety of different linkers were usedbetween the siRNA and PEG, after i.v administration of a single dose ofsiRNA measurable tumor tissue EGFR downregulation was achieved relativeto the negative control siRNA sequence or PBS controls. In addition, asillustrated in FIG. 80B, the different linker configurations resulted intumor siRNA accumulation at higher levels than the other tissue samplesmeasured (liver, spleen, lung and kidney).

In this example, it was demonstrated biological activity with a range ofA-X—B—Y—C conjugates in which a variety of different linkers strategies(component Y) were used to conjugate the PEG to the siRNA passengerstrand.

Example 38: 2016-PK-198-HCC827

siRNA Design and Synthesis

EFGR: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against human EGFR. The sequence ofthe guide/antisense strand was complementary to the gene sequencestarting a base position 333 for the human mRNA transcript for EGFR(ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base, sugar and phosphatemodifications were used to optimize the potency of the duplex and reduceimmunogenicity. All siRNA single strands were fully assembled on solidphase using standard phospharamidite chemistry and purified over HPLC.Purified single strands were duplexed to get the double stranded siRNA.The passenger strand contained two conjugation handles, a C6-NH₂ at the5′ end and a C6-SH at the 3′ end. Both conjugation handles wereconnected to siRNA passenger strand via phosphorothioate-invertedabasic-phosphorothioate linker, see Example 9 for the chemicalstructure.

Negative control siRNA sequence (scramble): A published (Burke et al.(2014) Pharm. Res., 31(12):3445-60) 21mer duplex with 19 bases ofcomplementarity and 3′ dinucleotide overhangs was used. The sequence (5′to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ IDNO: 2116). Base, sugar and phosphate modifications were used to reduceimmunogenicity and were comparable to those used in the active siRNA.All siRNA single strands were fully assembled on solid phase usingstandard phospharamidite chemistry and purified over HPLC. Purifiedsingle strands were duplexed to get the double stranded siRNA. Thepassenger strand contained two conjugation handles, a C6-NH₂ at the 5′end and a C6-SH at the 3′ end. Both conjugation handles were connectedto siRNA passenger strand via a phosphodiester-invertedabasic-phosphodiester linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASCarchitecture-4, as described in Example 9.

In Vivo Study Design

Groups 1-15 (n=5) of female NCr nu/nu mice bearing subcutaneously (SC)flank HCC827 tumors 100-300 mm³ in volume were treated with oneintravenous (i.v.) tail vein injection of siRNA conjugate, while controlgroup 16 (n=5) of the same mice received one i.v. injection of PBS as avehicle control. Table 50 describes the study design. Mice weresacrificed by CO₂ asphyxiation at 96 hours post-dose. 50 mg pieces oftumor and liver, were collected and snap-frozen in liquid nitrogen. mRNAknockdown in target tissue was determined using a comparative qPCR assayas described in Example 2. Total RNA was extracted from the tissue,reverse transcribed and mRNA levels were quantified using TaqMan qPCR,using the appropriately designed primers and probes. PPIB (housekeepinggene) was used as an internal RNA loading control, results werecalculated by the comparative Ct method, where the difference betweenthe target gene Ct value and the PPIB Ct value (ΔCt) is calculated andthen further normalized relative to the PBS control group by taking asecond difference (ΔΔCt). Quantitation of tissue siRNA concentrationswere determined using a stem-loop qPCR assay as described in Example 2.The antisense strand of the siRNA was reverse transcribed using a TaqManMicroRNA reverse transcription kit using a sequence-specific stem-loopRT primer. The cDNA from the RT step was then utilized for real-time PCRand Ct values were transformed into plasma or tissue concentrationsusing the linear equations derived from the standard curves.

TABLE 50 siRNA Dose Harvest Dose Volume # of Time Group Test Article N(mg/kg) ROA (mL/kg) Doses (h) 1 EGFR-Ab(Cys)-EGFR- 5 1 IV 5.0 1 96 PEG2k(n = 1) 2 EGFR-Ab(Cys)-EGFR- 5 0.5 IV 5.0 1 96 PEG2k (n = 1) 3EGFR-Ab(Cys)-EGFR- 5 0.25 IV 5.0 1 96 PEG2k (n = 1) 4 EGFR-Ab(Cys)-EGFR-5 1 IV 5.0 1 96 dPEG₄₈ (n = 1) 5 EGFR-Ab(Cys)-EGFR- 5 0.5 IV 5.0 1 96dPEG₄₈ (n = 1) 6 EGFR-Ab(Cys)-EGFR- 5 0.25 IV 5.0 1 96 dPEG₄₈ (n = 1) 7EGFR-Ab(Cys)-EGFR- 5 1 IV 5.0 1 96 dPEG₂₄ (n = 1) 8 EGFR-Ab(Cys)-EGFR- 50.5 IV 5.0 1 96 dPEG₂₄ (n = 1) 9 EGFR-Ab(Cys)-EGFR- 5 0.25 IV 5.0 1 96dPEG₂₄ (n = 1) 10 EGFR-Ab(Cys)-EGFR- 5 1 IV 5.0 1 96 dPEG₁₂ (n = 1) 11EGFR-Ab(Cys)-EGFR- 5 0.5 IV 5.0 1 96 dPEG₁₂ (n = 1) 12EGFR-Ab(Cys)-EGFR- 5 0.25 IV 5.0 1 96 dPEG₁₂ (n = 1) 13EGFR-Ab(Cys)-EGFR- 5 1 IV 5.0 1 96 PEG5k (n = 1) 14 PSMA-Ab(Cys)-EGFR- 51 IV 5.0 1 96 PEG5k (n = 1) 15 EGFR-Ab(Cys)-scramble- 5 1 IV 5.0 1 96PEG5k (n = 1) 16 PBS Control 5 — IV 5.0 1 96 Total # of Animals: 80nu/nu mice with HCC827 tumors

As illustrated in FIG. 81A, all the ASC with the differentconfigurations of linear PEG length achieved dose dependent EGFR mRNAknockdown in the HCC827 tumor cells, relative to the negative controlsiRNA sequence (scramble) and PBS controls. As illustrated in FIG. 81B,all the ASC with the different configurations in linear PEG lengthachieved equivalent dose dependent siRNA tumor tissue accumulation. Inaddition to low liver, lung, kidney and spleen accumulation relative totumor.

In this example, it was demonstrated biological activity with a range ofA-X—B—Y—C conjugates in which a variety of different PEG (component C)lengths were used.

Example 39: 2016-PK-194-WT

siRNA Design and Synthesis

EFGR: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against human EGFR. The sequence ofthe guide/antisense strand was complementary to the gene sequencestarting a base position 333 for the human mRNA transcript for EGFR(ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base, sugar and phosphatemodifications were used to optimize the potency of the duplex and reduceimmunogenicity. All siRNA single strands were fully assembled on solidphase using standard phospharamidite chemistry and purified over HPLC.Purified single strands were duplexed to get the double stranded siRNA.The passenger strand contained two conjugation handles, a C6-NH₂ at the5′ end and a C6-SH at the 3′ end. Both conjugation handles wereconnected to siRNA passenger strand via phosphorothioate-invertedabasic-phosphorothioate linker, see Example 9 for the chemicalstructure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASCarchitecture-4, as described in Example 9.

In Vivo Study Design

Groups (n=4) of wild-type female CD-1 mice were treated with oneintravenous (i.v.) tail vein injections of siRNA conjugates. Treatmentgroups received 0.5 mg/kg (based on the weight of siRNA) and all groupswere administered a dose volume of 5.0 mL/kg. Table 51 illustrates thestudy design in more detail. Non-terminal blood samples were collectedat 5, 30, and 180 minutes post-dose via puncture of the retro-orbitalplexus and centrifuged to generate plasma for PK analysis. Mice weresacrificed by CO₂ asphyxiation at 24, 96, or 168 h post-dose. Terminalblood samples were collected via cardiac puncture and processed togenerate plasma for PK analysis. Quantitation of plasma siRNAconcentrations were determined using a stem-loop qPCR assay as describedin Example 2. The antisense strand of the siRNA was reverse transcribedusing a TaqMan MicroRNA reverse transcription kit using asequence-specific stem-loop RT primer. The cDNA from the RT step wasthen utilized for real-time PCR and Ct values were transformed intoplasma or tissue concentrations using the linear equations derived fromthe standard curves.

TABLE 51 Dose Survival Terminal siRNA Dose Volume # of Bleed Bleed GroupTest Article N (mg/kg) ROA (mL/kg) Doses (min) (h) 1 EGFR-Ab(Cys)- 4 0.5IV 5.0 1 5 24 2 EGFR-PEG2k (n = 1) 4 0.5 IV 5.0 1 30 96 3 4 0.5 IV 5.0 1180 168 4 EGFR-Ab(Cys)- 4 0.5 IV 5.0 1 5 24 5 EGFR-dPEG₄₈ (n = 1) 4 0.5IV 5.0 1 30 96 6 4 0.5 IV 5.0 1 180 168 7 EGFR-Ab(Cys)- 4 0.5 IV 5.0 1 524 8 EGFR-dPEG₂₄ (n = 1) 4 0.5 IV 5.0 1 30 96 9 4 0.5 IV 5.0 1 180 16810 EGFR-Ab(Cys)- 4 0.5 IV 5.0 1 5 24 11 EGFR-dPEG₁₂ (n = 1) 4 0.5 IV 5.01 30 96 12 4 0.5 IV 5.0 1 180 168 Total # of Animals: 48 WT mice CD-1

As illustrated on slide 54, all the ASC with the different linear PEGlengths achieved equivalent plasma PK profiles, with approximately 10%of the siRNA remaining 168 hours after administration.

In this example, it was demonstrated equivalent plasma PK propertieswith a range of A-X—B—Y—C conjugates in which a variety of different PEG(component C) lengths were used.

Example 40: 2016-PK-195-WT

siRNA Design and Synthesis

EFGR: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against human EGFR. The sequence ofthe guide/antisense strand was complementary to the gene sequencestarting a base position 333 for the human mRNA transcript for EGFR(ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base, sugar and phosphatemodifications were used to optimize the potency of the duplex and reduceimmunogenicity. All siRNA single strands were fully assembled on solidphase using standard phospharamidite chemistry and purified over HPLC.Purified single strands were duplexed to get the double stranded siRNA.The passenger strand contained two conjugation handles, a C6-NH2 at the5′ end and a C6-SH at the 3′ end. Both conjugation handles wereconnected to siRNA passenger strand via phosphorothioate-invertedabasic-phosphorothioate linker, see Example 9 for the chemicalstructure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASCarchitecture-4, as described in Example 9.

In Vivo Study Design

Groups (n=4) of wild-type female CD-1 mice were treated with oneintravenous (i.v.) tail vein injections of siRNA conjugates. Treatmentgroups received 0.5 mg/kg (based on the weight of siRNA) and all groupswere administered a dose volume of 5.0 mL/kg. Table 52 illustrates thestudy design in more detail. Non-terminal blood samples were collectedat 5, 30, and 180 minutes post-dose via puncture of the retro-orbitalplexus and centrifuged to generate plasma for PK analysis. Mice weresacrificed by CO₂ asphyxiation at 24, 96, or 168 h post-dose. Terminalblood samples were collected via cardiac puncture and processed togenerate plasma for PK analysis. Quantitation of plasma siRNAconcentrations were determined using a stem-loop qPCR assay as describedin Example 2. The antisense strand of the siRNA was reverse transcribedusing a TaqMan MicroRNA reverse transcription kit using asequence-specific stem-loop RT primer. The cDNA from the RT step wasthen utilized for real-time PCR and Ct values were transformed intoplasma or tissue concentrations using the linear equations derived fromthe standard curves.

TABLE 52 siRNA Dose Survival Terminal Dose Volume # of Bleed Bleed GrTest Article N (mg/kg) ROA (mL/kg) Doses (min) (h) 1EGFR-Ab(Cys)-EGFR-PEG10k 4 0.5 IV 5.0 1 5 24 2 (n = 1) 4 0.5 IV 5.0 1 3096 3 4 0.5 IV 5.0 1 180 168 4 EGFR-Ab(Cys)-EGFR- 4 0.5 IV 5.0 1 5 24 5(dPEG24)₃ (n = 1) 4 0.5 IV 5.0 1 30 96 6 4 0.5 IV 5.0 1 180 168 7EGFR-Ab(Cys)-EGFR- 4 0.5 IV 5.0 1 5 24 8 (dPEG12)₃ (n = 1) 4 0.5 IV 5.01 30 96 9 4 0.5 IV 5.0 1 180 168 10 EGFR-Ab(Cys)-EGFR-(dPEG4)₃ 4 0.5 IV5.0 1 5 24 11 (n = 1) 4 0.5 IV 5.0 1 30 96 12 4 0.5 IV 5.0 1 180 168Total # of Animals: 48 WT mice CD-1

As illustrated in FIG. 83, all the ASC with the different PEGconfigurations (length and branching) achieved equivalent plasma PKprofiles, with approximately 10% of the siRNA remaining 168 hours afteradministration.

In this example, it was demonstrated equivalent plasma PK propertieswith a range of A-X—B—Y—C conjugates in which a variety of different PEG(component C) lengths and branching were used.

Example 41: 2016-PK-236-HCC827

siRNA Design and Synthesis

EFGR: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against human EGFR. The sequence ofthe guide/antisense strand was complementary to the gene sequencestarting a base position 333 for the human mRNA transcript for EGFR(ACUCGUGCCUUGGCAAACUUU; SEQ ID NO: 2082). Base, sugar and phosphatemodifications were used to optimize the potency of the duplex and reduceimmunogenicity. All siRNA single strands were fully assembled on solidphase using standard phospharamidite chemistry and purified over HPLC.Purified single strands were duplexed to get the double stranded siRNA.The passenger strand contained two conjugation handles, a C6-NH₂ at the5′ end and a C6-SH at the 3′ end. Both conjugation handles wereconnected to siRNA passenger strand via phosphorothioate-invertedabasic-phosphorothioate linker, see Example 9 for the chemicalstructure.

Negative control siRNA sequence (scramble): A published (Burke et al.(2014) Pharm. Res., 31(12):3445-60) 21mer duplex with 19 bases ofcomplementarity and 3′ dinucleotide overhangs was used. The sequence (5′to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ IDNO: 2116). Base, sugar and phosphate modifications were used to reduceimmunogenicity and were comparable to those used in the active siRNA.All siRNA single strands were fully assembled on solid phase usingstandard phospharamidite chemistry and purified over HPLC. Purifiedsingle strands were duplexed to get the double stranded siRNA. Thepassenger strand contained two conjugation handles, a C6-NH₂ at the 5′end and a C6-SH at the 3′ end. Both conjugation handles were connectedto siRNA passenger strand via a phosphodiester-invertedabasic-phosphodiester linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

All conjugates were made and purified as a DAR1 (n=1) using ASCarchitecture-4, as described in Example 9.

In Vivo Study Design

Groups 1-12 (n=5) of female NCr nu/nu mice bearing subcutaneously (SC)flank HCC827 tumors 100-300 mm³ in volume were treated with oneintravenous (i.v.) tail vein injection of siRNA conjugate, while controlgroup 13 (n=5) of the same mice received one i.v. injection of PBS as avehicle control. Table 53 describes the study design. Mice weresacrificed by CO₂ asphyxiation at 96 hours post-dose. 50 mg pieces oftumor and liver, were collected and snap-frozen in liquid nitrogen. mRNAknockdown in target tissue was determined using a comparative qPCR assayas described in Example 2. Total RNA was extracted from the tissue,reverse transcribed and mRNA levels were quantified using TaqMan qPCR,using the appropriately designed primers and probes. PPIB (housekeepinggene) was used as an internal RNA loading control, results werecalculated by the comparative Ct method, where the difference betweenthe target gene Ct value and the PPIB Ct value (ΔCt) is calculated andthen further normalized relative to the PBS control group by taking asecond difference (ΔΔCt). Quantitation of tissue siRNA concentrationswere determined using a stem-loop qPCR assay as described in Example 2.The antisense strand of the siRNA was reverse transcribed using a TaqManMicroRNA reverse transcription kit using a sequence-specific stem-loopRT primer. The cDNA from the RT step was then utilized for real-time PCRand Ct values were transformed into plasma or tissue concentrationsusing the linear equations derived from the standard curves.

TABLE 53 siRNA Dose Harvest Dose Volume # of Time Group Test Article N(mg/kg) ROA (mL/kg) Doses (h) 1 EGFR-Ab(Cys)-EGFR-PEG10k (n = 1) 5 1 IV5.0 1 96 2 EGFR-Ab(Cys)-EGFR-PEG10k (n = 1) 5 0.5 IV 5.0 1 96 3EGFR-Ab(Cys)-EGFR-PEG10k (n = 1) 5 0.25 IV 5.0 1 96 4EGFR-Ab(Cys)-EGFR-(dPEG24)3 (n = 1) 5 1 IV 5.0 1 96 5EGFR-Ab(Cys)-EGFR-(dPEG24)3 (n = 1) 5 0.5 IV 5.0 1 96 6EGFR-Ab(Cys)-EGFR-(dPEG24)3 (n = 1) 5 0.25 IV 5.0 1 96 7EGFR-Ab(Cys)-EGFR-(dPEG12)3 (n = 1) 5 1 IV 5.0 1 96 8EGFR-Ab(Cys)-EGFR-(dPEG12)3 (n = 1) 5 0.5 IV 5.0 1 96 9EGFR-Ab(Cys)-EGFR-(dPEG12)3 (n = 1) 5 0.25 IV 5.0 1 96 10EGFR-Ab(Cys)-EGFR-(dPEG4)3 (n = 1) 5 1 IV 5.0 1 96 11EGFR-Ab(Cys)-EGFR-PEG5k (n = 1) 5 1 IV 5.0 1 96 12EGFR-Ab(Cys)-scramble-PEG5k (n = 1) 5 1 IV 5.0 1 96 13 PBS Control 5 —IV 5.0 1 96 Total # of Animals: 65 nu/nu mice with HCC827 tumors

As illustrated in FIG. 84, all the ASC with the different configurationsof PEG (length and branching) achieved equivalent EGFR mRNA knockdown inthe HCC827 tumor cells to the construct with the linear PEG5K at the 1mg/kg dose. Those constructs tested in a dose response format, showeddose dependent knockdown of EGFR mRNA. As illustrated in FIG. 85, allthe ASC with the different variations in linear PEG length and PEGbranching achieved equivalent siRNA tumor tissue accumulation to theconstruct with the linear PEG5K at the 1 mg/kg dose. In addition to lowliver accumulation relative to tumor, those constructs tested in a doseresponse format, showed dose dependent tumor tissue accumulation ofsiRNA.

In this example, it was demonstrated biological activity with a range ofA-X—B—Y—C conjugates in which a variety of different PEG (component C)lengths and branching were used.

Example 42: In Vitro Knockdown with ASCs with PEG Polymers

siRNA Design and Synthesis

HPRT: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against human HPRT. The sequence ofthe guide/antisense strand was AUAAAAUCUACAGUCAUAGUU (SEQ ID NO: 2082)and design to be complementary to the gene sequence starting a baseposition 425 for the human mRNA transcript for HPRT. Base, sugar andphosphate modifications were used to optimize the potency of the duplexand reduce immunogenicity. All siRNA single strands were fully assembledon solid phase using standard phospharamidite chemistry and purifiedover HPLC. Purified single strands were duplexed to get the doublestranded siRNA. The passenger strand contained two conjugation handles,a C6-NH₂ at the 5′ end and a C6-SH at the 3′ end. Both conjugationhandles were connected to siRNA passenger strand viaphosphodiester-inverted abasic-phosphorothioate linker. The C6-NH2 andC6-SH were connected through the phosphodiester, see Example 9 for thechemical structure.

Negative control siRNA sequence (scramble): A published (Burke et al.(2014) Pharm. Res., 31(12):3445-60) 21mer duplex with 19 bases ofcomplementarity and 3′ dinucleotide overhangs was used. The sequence (5′to 3′) of the guide/antisense strand was UAUCGACGUGUCCAGCUAGUU (SEQ IDNO: 2116). The same base, sugar and phosphate modifications that wereused for the active EGFR siRNA duplex were used in the negative controlsiRNA. All siRNA single strands were fully assembled on solid phaseusing standard phospharamidite chemistry and purified over HPLC.Purified single strands were duplexed to get the double stranded siRNA.The passenger strand contained two conjugation handles, a C6-NH₂ at the5′ end and a C6-SH at the 3′ end. Both conjugation handles wereconnected to siRNA passenger strand via a phosphodiester-invertedabasic-phosphodiester linker, see Example 9 for the chemical structure.

ASC Synthesis and Characterization

Conjugates in groups 1-3 made and purified as a DAR1 (n=1) using ASCarchitecture-1, as described in Example 9. Conjugates in groups 4-6 weremade and purified as a DAR1 (n=1) using ASC architecture-4, as describedin Example 9.

In Vitro Study Design

Mouse spleens were harvested and kept in PBS with 100 u/ml penicillinand streptomycin on ice. Spleens were smashed with clean glass slides,cut into small pieces, homogenized with 18G needles, and filtered (70 umnylon membrane). Dead cells were removed with the dead cell removal kitfrom Milteny biotec (Catalog #130-090101) according to manufacturerinstruction. To isolate mouse B cells, B cell isolation kit Miltenybiotec (Catalog #130-090-862) was used following manufacturerinstruction. Briefly, live spleen cells were resuspended with 200 μl ofMACS buffer per mouse spleen. Non-B cells were depleted withbiotin-conjugated monoclonal antibodies against CD43 (Ly48), CD4, andTer-119, coupled with anti-biotin magnetic microbeads. From one mousespleen, 30 million live B cells can be obtained. To activate isolatedmouse B cells (2×10⁶/ml in 10% FBS RPMI-1640 with 100 u/ml penicillinand streptomycin), a cocktail of 10 μg/ml LPS, 5 μg/ml anti-IgM, 1 μg/mlanti-CD40, 0.05 μg/ml IL-4, and 0.05 μg/ml INFγ was added. After fourhours of activation, ASCs (1 pM to 10 nM) were added to 10⁶ cells perwell in 24 (0.5 ml media) or 12 (1 ml media) well plates. After 48 hoursof ASC treatments, cells were harvested and isolated RNAs were analyzedfor mRNA knockdown. See Table 54 for the study design.

TABLE 54 Group Test Article 1 Anti-B cell Ab(Lys)-S3′-HPRT-5′N-pOEGMA8K2 Anti-B cell Ab(Lys)-S3′-HPRT-5′N-pHPMA5K 3 Anti-B cellAb(Lys)-S3′-HPRT-5′N-pHPMA10K 4 Anti-B cell Ab(Cys)-N5′-HPRT-3′S-pMAA10K5 Anti-B cell Ab(Cys)-N5′-HPRT-3′S-PEG5K 6 Anti-B cellAb(Cys)-N5′-scramble-3′S-PEG5K

In this in vitro experiment in activated primary mouse B cells, theability of an anti-B cell antibody ASCs to deliver an siRNA design todownregulate Hypoxanthine-guanine phosphoribosyltransferase (HPRT) witha range of alternative PEG polymers were measured. As illustrated inFIG. 86, the range of ASC with alternative PEGs were able todownregulate HPRT relative to the scramble control.

In this example, the biological activity was demonstrated with a rangeof A-X—B—Y—C conjugates in which a variety of polymer alternatives toPEG (component C) were used.

Example 43: PK-236-WT

siRNA Design and Synthesis

KRAS: A 21mer duplex with 19 bases of complementarity and 3′dinucleotide overhangs was designed against human KRAS. The sequence ofthe guide/antisense strand was complementary to the gene sequencestarting a base position 237 for the human mRNA transcript for KRAS(Guide strand sequence: UGAAUUAGCUGUAUCGUCAUU; SEQ ID NO: 2088). Base,sugar and phosphate modifications that are well described in the fieldof RNAi were used to optimize the potency of the duplex and reduceimmunogenicity. All siRNA single strands were fully assembled on solidphase using standard phospharamidite chemistry and purified over HPLC.The base at position 11 on the passenger strand had a Cy5 fluorescentlabel attached, as described in Example 9. Purified single strands wereduplexed to get the double stranded siRNA. The passenger strandcontained two conjugation handles, a C6-NH₂ at the 5′ end and a C6-SH atthe 3′ end. Both conjugation handles were connected to siRNA passengerstrand via a phosphodiester-inverted abasic-phosphodiester linker, seeExample 9 for the chemical structure.

ASC Synthesis and Characterization

Conjugates in groups 1-3 were made and purified as a DAR1 (n=1) usingASC architecture-4, as described in Example 9. Conjugates in groups 4-6were made and purified as a DAR1 (n=1) using ASC architecture-4, butthere was no PEG on the 3′ end of the passenger strand. Prior toconjugateion, the 3′thiol was end-capped using N-ethylmaleimide.Conjugates in groups 7-9 were made and purified as a DAR1 (n=1) usingASC architecture-1, as described in Example 9. Conjugates in groups10-12 made and purified as a DAR1 (n=1) using ASC architecture-1, butthere was no PEG on the 5′ end of the passenger strand.

In Vivo Study Design

Groups (n=4) of wild-type female CD-1 mice were treated with oneintravenous (i.v.) tail vein injections of siRNA conjugates. Treatmentgroups received 0.5 mg/kg (based on the weight of siRNA) and all groupswere administered a dose volume of 5.0 mL/kg. Table 55 illustrates thestudy design in more detail. Non-terminal blood samples were collectedat 0.25, 1, and 4 hours post-dose via puncture of the retro-orbitalplexus and centrifuged to generate plasma for PK analysis. Mice weresacrificed by CO₂ asphyxiation at 24, 48, or 72 h post-dose. Terminalblood samples were collected via cardiac puncture and processed togenerate plasma for PK analysis.

Plasma samples (K2 EDTA) were processed within 4 hours after harvesting.Plasma samples were diluted with matching mouse plasma (Bioreclamation)(2-400 fold) and the concentration of CY5-siRNA in these plasma samplesquantified spectroscopically using a TECAN Infinite M200 Pro (Excitation635 nm; Emission 675 nm). To release macromolecular interactions thatmight quench the CY5 fluorescence, all samples were diluted 2-fold intowater containing 0.01% Tween 20 and 100 ug/ml heparin prior toquantification. To determine the amount of intact ASCs in these plasmasamples, plasma samples were diluted with mouse plasma to 2-50 nMCY5-siRNA and incubated with Protein G Dynabeads (Thermofisher) loadedwith 150 nM of a purified EGFR-Fc protein (Sino Biological). Thesebinding reactions were incubated at RT for 1 hour. Beads were washedtwice with PBS containing 0.01% Tween 20 and 0.05% BSA before ASCs boundto EGFR were eluted by incubation in 0.1 M citric acid (pH 2.7). Theamount of CY5-siRNA contained in the input, unbound fraction, washes andbead eluate was quantified by fluorescence as stated above.

TABLE 55 siRNA Dose Survival Terminal Dose Volume # of Bleed Bleed GrTest Article N (mg/kg) ROA (mL/kg) Doses (h) (h) 1EGFR-Ab(Cys)-N5′-Cy5.KRAS- 4 0.5 IV 5.0 1 0.25 24 3′S-PEG5k (n = 1) 2EGFR-Ab(Cys)-N5′-Cy5.KRAS- 4 0.5 IV 5.0 1 1 48 3′S-PEG5k (n = 1) 3EGFR-Ab(Cys)-N5′-Cy5.KRAS- 4 0.5 IV 5.0 1 4 72 3′S-PEG5k (n = 1) 4EGFR-Ab(Cys)-N5′-Cy5.KRAS- 4 0.5 IV 5.0 1 0.25 24 3′S--NEM (n = 1) 5EGFR-Ab(Cys)-N5′-Cy5.KRAS- 4 0.5 IV 5.0 1 1 48 3′S--NEM (n = 1) 6EGFR-Ab(Cys)-N5′-Cy5.KRAS- 4 0.5 IV 5.0 1 4 72 3′S--NEM (n = 1) 7EGFR-Ab(Lys)-S3′-Cy5.KRAS- 4 0.5 IV 5.0 1 0.25 24 5′N-PEG5k (n = 1) 8EGFR-Ab(Lys)-S3′-Cy5.KRAS- 4 0.5 IV 5.0 1 1 48 5′N-PEG5k (n = 1) 9EGFR-Ab(Lys)-S3′-Cy5.KRAS- 4 0.5 IV 5.0 1 4 72 5′N-PEG5k (n = 1) 10EGFR-Ab(Lys)-S3′-Cy5.KRAS- 4 0.5 IV 5.0 1 0.25 24 5′NH₂ (n = 1) 11EGFR-Ab(Lys)-S3′-Cy5.KRAS- 4 0.5 IV 5.0 1 1 48 5′NH₂ (n = 1) 12EGFR-Ab(Lys)-S3′-Cy5.KRAS- 4 0.5 IV 5.0 1 4 72 5′NH₂ (n = 1) Total # ofAnimals: 96 WT mice CD-1

In this in vivo PK study, the in vivo plasma stability of two AXBYCconjugates (cysteine and lysine conjugation to the EGFR-Ab) relative totwo AXB conjugates were compared. As illustrated in FIG. 87, theconcentration of the siRNA was determined using two methods. Thefluorescence of the plasma was measured directly and the siRNAconcentration determined using a standard curve. Or a magnetic beaddecorated with EGFR was used to bind the antibody conjugates and thenthe fluorescence of the sample was measured and the siRNA concentrationdetermined using a standard curve. All data were plotted as a percentageof the injected dose. In both examples of the AXBYC conjugates (cysteineand lysine conjugation to the EGFR-Ab) improved plasma PK were observedrelative to the corresponding AXB conjugate.

In this example, in vivo plasma PK for the Cys and Lys AXBYC conjugatescompared to the matching control AXB conjugates was demonstrate.

Example 44: In Vivo Pharmacodynamics Study of a Cholesterol-KRASConjugate (PD-058)

Groups (n=5) of female NCr nu/nu mice bearing intrahepatic Hep 3B tumorsone week after inoculation were treated with three intravenous (i.v.)tail vein injections (separated by 48 h) of cholesterol-siRNA conjugate,while control groups (n=5) of the same mice received three i.v.injections of PBS as a vehicle control on the same dosing schedule.Treatment groups that received chol-KRAS were dosed at 10, 4, or 2mg/kg. All groups (treatments and controls) were administered a dosevolume of 6.25 mL/kg. Table 56 describes the study design in more detailand gives a cross-reference to the conjugate synthesis andcharacterization. Mice were sacrificed by CO₂ asphyxiation at 72 hpost-final dose. 50 mg pieces of tumor-bearing liver were collected andsnap-frozen in liquid nitrogen. mRNA knockdown analysis and siRNAquantitation were performed as described in Examples 2-7.

TABLE 56 Study design for a Cholesterol-KRAS Conjugate (PD-058) with across-reference to the synthesis and characterization of the conjugatestested. siRNA Dose Cross-reference for synthesis and Group Test ArticleN (mg/kg) ROA # of Doses characterization 1 Chol-KRAS 5 10 iv 3 Generalexperimental (Example 2) 2 Chol-KRAS 5 4 iv 3 General experimental(Example 2) 3 Chol-KRAS 5 2 iv 3 General experimental (Example 2) 4Vehicle (PBS) 5 iv 3

The chol-KRAS conjugate was assessed for mRNA knockdown in a 3-dosestudy with a dose response. As illustrated in FIG. 35, within the mouseliver tissue there was a clear dose-response for mouse KRAS mRNAknockdown. The lowest dose of 2 mg/kg resulted in 45% knockdown of mouseKRAS, while the highest dose of 10 mg/kg resulted in 65% knockdown ofmouse KRAS in this 3-dose format. However, there were not enough humantumor cells in the mouse liver at the time of harvest to detect a signalfrom human KRAS (potentially due to model development issues, itappeared that not enough human cells were inoculated to producefast-growing tumors). As such, it was not possible to measure theknockdown in tumor.

Example 45: In Vivo Pharmacokinetics Study of a Cholesterol-siRNAConjugate (PK-063)

Groups (n=3) of wild-type female CD-1 mice were treated with either oneor two intravenous (i.v.) tail vein injections of chol-siRNA conjugate.Treatment groups received chol-KRAS at 10 mg/kg (based on the weight ofsiRNA) and the 2-dose groups received the second dose 48 h after thefirst dose. All groups were administered a dose volume of 6.25 mL/kg.Table 57 illustrates the study design in more detail and gives across-reference to the conjugate synthesis and characterization.Non-terminal blood samples were collected at 2, 15, 60 or 120 minutespost-final dose via puncture of the retro-orbital plexus and centrifugedto generate plasma for PK analysis. Mice were sacrificed by CO₂asphyxiation at 4, 24, 96, or 144 h post-final dose. Terminal bloodsamples were collected via cardiac puncture and processed to generateplasma for PK analysis. 50 mg pieces of tumor, liver, kidney, and lungwere collected and snap-frozen in liquid nitrogen. mRNA knockdownanalysis and siRNA quantitation were performed as described in Examples2-7.

TABLE 57 Study design for a Cholesterol-siRNA Conjugate (PK-063) with across-reference to the synthesis and characterization of the conjugatestested. siRNA Survival Terminal Harvest Cross-reference Test Dose # ofBleed Bleed Time to synthesis and Group Article N (mg/kg) ROA Doses(min) (h) (h) characterization 1 Chol- 3 10 IV 1 2 4 4 General 2 KRAS 310 IV 1 15 24 24 experimental 3 3 10 IV 1 60 96 96 (Example 2) 4 3 10 IV1 120 144 144 5 Chol- 3 10 IV 2 2 4 4 General 6 KRAS 3 10 IV 2 15 24 24experimental 7 3 10 IV 2 60 96 96 (Example 2) 8 3 10 IV 2 120 144 144

The pharmacokinetic behavior of chol-siRNA was assessed in a single-doseformat compared to a 2-dose format. As illustrated from FIG. 36, theplasma PK profiles for the first dose and a second dose following 48 hlater are nearly identical. The mechanism for clearance from plasma hasnot saturated from the first dose and the second dose behaves similarly.The tissue PK for 3 major tissues (the liver, kidneys, and lungs) wassimilarly assessed. As illustrated from FIG. 37, chol-KRAS was deliveredto liver in the highest concentrations, with kidneys and lungs havingapproximately 10-fold lower siRNA concentrations compared to liver. Forall three tissues, the siRNA concentrations following the second doseswere higher than the siRNA concentrations following the first dose,demonstrating that there is accumulation of siRNA in tissues when dosesof chol-siRNA are spaced by 48 h.

In Vivo Study a Cholesterol-siRNA Conjugate (PK-067).

Groups (n=3) of female NCr nu/nu mice bearing subcutaneous flank H358tumors 100-150 mm³ in volume were treated with one intravenous (i.v.)tail vein injection of siRNA conjugate, while control groups (n=4) ofthe same mice received one i.v. injection of PBS as a vehicle control.Treatment groups that received cholesterol-siRNA conjugates were dosedat 5 mg/kg (based on the weight of siRNA). Some treatment groups alsoreceived cholesterol-peptide conjugates at specified molar peptide:siRNAratios, where all chol-siRNA and chol-peptide conjugates were mixedtogether in solution and co-injected. All groups (treatments andcontrols) were administered a dose volume of 5 mL/kg. Table 58 shows thestudy design in more detail and gives a cross-reference to the conjugatesynthesis and characterization. Mice were sacrificed by CO₂ asphyxiationat 24, 72, or 144 h post-dose. 50 mg pieces of tumor, liver, kidneys,and lungs were collected and snap-frozen in liquid nitrogen. mRNAknockdown analysis and siRNA quantitation were performed as described inExamples 2-7.

TABLE 58 Study design for a Cholesterol-siRNA Conjugate (PK-067) with across-reference to the conjugate synthesis and characterization molsiRNA EEP/mol Harvest Cross-reference Dose siRNA # of Time to synthesisand Group Test Article N (mg/kg) Ratio ROA Doses (h) characterization 1chol-KRAS 3 5 — IV 1 24 General 2 3 5 — IV 1 72 experimental 3 3 5 — IV1 144 (Example 2) 4 chol-KRAS + 3 5 1 IV 1 24 General 5 chol-Melittin 35 1 IV 1 72 experimental 6 3 5 1 IV 1 144 (Example 2) 7 chol-KRAS + 3 53 IV 1 24 General 8 chol-Melittin 3 5 3 IV 1 72 experimental 9 3 5 3 IV1 144 (Example 2) 10 chol-KRAS + 3 5 10 IV 1 24 General 11 chol-Melittin3 5 10 IV 1 72 experimental 12 3 5 10 IV 1 144 (Example 2) 13chol-KRAS + 3 5 1 IV 1 24 General 14 chol-INF7 3 5 1 IV 1 72experimental 15 3 5 1 IV 1 144 (Example 2) 16 chol-KRAS + 3 5 3 IV 1 24General 17 chol-INF7 3 5 3 IV 1 72 experimental 18 3 5 3 IV 1 144(Example 2) 19 chol-KRAS + 3 5 10 IV 1 24 General 20 chol-INF7 3 5 10 IV1 72 experimental 21 3 5 10 IV 1 144 (Example 2) 22 Vehicle (PBS) 4 — —IV 1 24 General 23 4 — — IV 1 72 experimental 24 4 — — IV 1 144 (Example2) Total # of 75 Animals: nu/nu mice with H358 tumors

Endosomolytic moieties (EEPs) such as INF7 and melittin were conjugatedto cholesterol, mixed with chol-siRNA, and then co-injected into mice todemonstrate an increase in siRNA potency due to the improved endosomalescape. First, the effect of adding the EEPs on the siRNA concentrationin various tissues was assessed. As illustrated in FIG. 38A, theaddition of chol-INF7 at any of the molar ratios of EEP:siRNA did notaffect the siRNA tumor PK. However, as illustrated in FIG. 38B, theaddition of chol-melittin at a 1:1 ratio did not affect the tumor PK butthe addition of chol-melittin at a 3:1 EEP:siRNA ratio decreased theamount of siRNA in tumor. As illustrated in FIG. 39, neither chol-INF7nor chol-melittin had much of an impact on the liver PK. Similarly, asillustrated in FIGS. 40 and 41, the chol-INF7 and chol-melittin also didnot have much of an impact on the PK profile in kidneys and lungs.Finally, the effect of the chol-EEP conjugates on mRNA KD was assessedand, as shown in FIG. 42, the baseline level of knockdown for chol-KRASalone was approximately 50%. The addition of 1:1 chol-melittin or 3:1chol-INF7 improves the knockdown at each time point, due to improvedendosomal escape.

In Vivo Study a Cholesterol-siRNA Conjugate (PK-076).

Groups (n=5) of female NCr nu/nu mice bearing subcutaneous flank H358tumors 100-150 mm³ in volume were treated with three intravenous (i.v.)tail vein injections of siRNA conjugate separated by 48 h, while controlgroups (n=5) of the same mice received three i.v. injections of PBS as avehicle control on the same dosing schedule. Treatment groups thatreceived cholesterol-siRNA conjugates were dosed at 5 mg/kg (based onthe weight of siRNA). Some treatment groups also receivedcholesterol-peptide conjugates at specified molar peptide:siRNA ratios,where all chol-siRNA and chol-peptide conjugates were mixed together insolution and co-injected. All groups (treatments and controls) wereadministered a dose volume of 5 mL/kg. Table 59 describes the studydesign in more detail and gives a cross-reference to the conjugatesynthesis and characterization. Mice were sacrificed by CO₂ asphyxiationat 24 or 96 h post-dose. 50 mg pieces of tumor, liver, kidneys, andlungs were collected and snap-frozen in liquid nitrogen. mRNA knockdownanalysis and siRNA quantitation were performed as described in Examples2-7.

TABLE 59 Study design for a Cholesterol-siRNA Conjugate (PK-076) with across-reference to the synthesis and characterization of the conjugatestested. siRNA EEP/siRNA Harvest Cross-reference to Dose Ratio # of Timesynthesis and Group Test Article N (mg/kg) (mol/mol) ROA Doses (h)characterization 1 chol-KRAS 5 5 — IV 3 24 General 2 5 5 — IV 3 96experimental (Example 2) 3 chol-KRAS + 5 5 1 IV 3 24 General 4chol-melittin (1:1) 5 5 1 IV 3 96 experimental (Example 2) 5 chol-KRAS +5 5 3 IV 3 24 General 6 chol-INF7 (3:1) 5 5 3 IV 3 96 experimental(Example 2) 7 Vehicle 5 — — IV 3 24 8 5 — — IV 3 96

The activity seen in the single-dose study with chol-siRNA and chol-EEPwas followed up with a three dose study. The 3:1 ratio of EEP:siRNA wasselected for INF7, and the 1:1 ratio was selected for melittin. Asillustrated in FIG. 43 and FIG. 44, the addition of either chol-EEP tothe chol-siRNA does not seem to greatly affect the tissue PK followingthree doses. As for the knockdown, FIG. 45 shows that addition ofchol-melittin clearly improves tumor knockdown 24 h post-dose. It alsoshows that chol-melittin improves tumor knockdown at 96 h post-dose.

In Vivo Study a Cholesterol-siRNA Conjugate (PK-079).

Groups (n=5) of female NCr nu/nu mice bearing subcutaneous flank H358tumors 100-150 mm³ in volume were treated with one intravenous (i.v.)tail vein injection of siRNA conjugate, while control groups (n=5) ofthe same mice received one i.v. injection of PBS as a vehicle control.Treatment groups that received EGFR antibody-siRNA-PEG conjugates weredosed at 0.5 mg/kg (based on the weight of siRNA) and groups that alsoreceived EGFR antibody-melittin had the dose of EGFR-Ab matched betweenEGFR antibody-siRNA and EGFR antibody-melittin. All groups (treatmentsand controls) were administered a dose volume of 5 mL/kg. Table 60describes the study design in more detail and gives a cross-reference tothe conjugate synthesis and characterization. Mice were sacrificed byCO₂ asphyxiation at 96 h post-dose. 50 mg pieces of tumor, liver,kidney, and lung were collected and snap-frozen in liquid nitrogen. mRNAknockdown analysis and siRNA quantitation were performed as described inExamples 2-7.

TABLE 60 Study design for a Cholesterol-siRNA Conjugate (PK-079) with across-reference to the synthesis and characterization of the conjugatestested. siRNA siRNA:EGFR- melittin:siRNA Harvest Cross-reference Dose AbRatio Ratio # of Time to synthesis and Group Test Article N (mg/kg)(mol/mol) (mol/mol) ROA Doses (h) characterization 1 EGFR-Ab- 5 0.5 1 —IV 1 96 Example 4 PEG5k-EGFR 2 EGFR-Ab- 5 0.5 1 1:1 IV 1 96 Example 3and 6 PEG5k-EGFR + EGFR-Ab- melittin 3 EGFR-Ab-KRAS- 5 0.5 1 1:1 IV 1 96Example 3 and 6 PEG5k + EGFR-Ab- melittin 4 EGFR antibody 5 — — — IV 196 General Alone experimental (Example 2) 5 Vehicle 5 — — — IV 1 96

The PK/PD relationship for EGFR antibody-siRNA conjugates to deliversiRNA to tumor and produce mRNA knockdown in tumor was evaluated forreproducibility. As illustrated in FIG. 46, once again a single i.v.dose of 0.5 mg/kg of EGFR antibody-siRNA conjugate was able to deliverapproximate 100 nM concentrations of siRNA into tumor with bothconfigurations of the conjugate. The addition of EGFR antibody-melittindid not appear to impact the tissue PK. Out of the four tissuesanalyzed, tumor had the highest concentration and liver the secondhighest, with kidneys and lungs showing low uptake of siRNA. Asillustrated in FIG. 47, the strong siRNA delivery to tumor once againtranslated into approximately 50% knockdown of EGFR or KRAS in thetumors. Free EGFR-Ab, run as a control group, showed no mRNA knockdownas did the PBS control.

In Vivo Study a Cholesterol-siRNA Conjugate (PD-077).

Groups (n=11) of female NCr nu/nu mice bearing intrahepatic Hep3B tumorsone week after inoculation were treated with nine intravenous (i.v.) orsubcutaneous (s.c.) injections (TIW) of cholesterol-siRNA conjugate,while control groups (n=11) of the same mice received nine i.v. tailvein injections of PBS as a vehicle control (also dosed TIW). Treatmentgroups that received chol-CTNNB1 were dosed at 5 mg/kg. All groups(treatments and controls) were administered a dose volume of 6.25 mL/kg.Table 61 describes the study design in more detail and gives across-reference to the conjugate synthesis and characterization.Non-terminal blood samples were collected once per week via puncture ofthe retro-orbital plexus and processed to generate serum foralpha-Fetoprotein (AFP) measurement. Mice were sacrificed by CO₂asphyxiation at 24 h post-final dose. 50 mg pieces of tumor-bearingliver were collected and snap-frozen in liquid nitrogen. mRNA knockdownanalysis was performed as described above. AFP was quantified using theHuman alpha-Fetoprotein DuoSet ELISA kit (R&D Systems) according to themanufacturer's instructions.

TABLE 61 Study design for a Cholesterol-siRNA Conjugate (PK-077) with across-reference to the synthesis and characterization of the conjugatestested. siRNA Terminal Cross-reference Dose # of Survival Bleed tosynthesis and Group Test Article N (mg/kg) ROA Doses Bleed (h)characterization 5 Chol-CTNNB1 11 5 IV 9 Weekly 24 General experimental(Example 2) 8 Chol-CTNNB1 11 5 SC 9 Weekly 24 General experimental(Example 2) 11 Vehicle 11 IV 9 Weekly 24 Total # of Animals: 33

Since earlier studies demonstrated that it was possible for a singledose of chol-siRNA to generate knockdown in normal liver, it washypothesized that knockdown could be achieved in orthotopic liver tumorsas well. Mice were inoculated with intrahepatic Hep3B tumors that wereallowed to grow for one week post-inoculation, and then these mice wereadministered 5 mg/kg doses of chol-CTNNB1 (either i.v. or s.c.) threetimes a week for three weeks (9 total doses). As illustrated in FIG. 48,the chol-CTNNB1 dosed s.c. was able to produce >50% mRNA knockdown atthe harvest time point of 24 h post-final dose. In contrast, thechol-CTNNB1 siRNA that was dosed i.v. does not seem to show any mRNAknockdown at this time point (although some mice did not have anymeasurable human CTNNB1 signal, it was hard to determine if the loss ofsignal was related to knockdown or low tumor burden). The human Hep3Bcells are also known to secrete human alpha-Fetoprotein (AFP), and it isknown that the amount of secreted AFP correlates with the number ofHep3B cells. Thus, the concentration of AFP in serum is taken as amarker of tumor load in the mouse, and the increase in AFP over timecorrelates with tumor growth. As illustrated in FIG. 49, the chol-CTNNB1dosed s.c. markedly reduced the AFP levels in those mice, which providesevidence that the CTNNB1 mRNA knockdown led to the inhibition of tumorgrowth.

Example 46. Liver PK/PD Study

Female wild-type CD-1 mice will be dosed with chol-siRNA-EEP conjugatesat 5 mg/kg (based on the weight of siRNA). In these studies the siRNAused will be against the mouse Factor VII (FVII) such that FVIIknockdown can be determined by measuring the FVII protein levels inplasma. Multiple EEPs (endosomolytic moieties) will be used to determinethe peptide sequence that demonstrates optimal endosomal escape,resulting in the best knockdown of the FVII target gene relative to thecontrol.

Example 47. Tumor PK/PD Study

Female NCr nu/nu mice bearing subcutaneous flank H358 tumors will bedosed with EGFR antibody-siRNA-EEP conjugates at 0.5 mg/kg (based onsiRNA). Multiple EEPs (endosomolytic moieties) will be used to determinethe peptide sequence that demonstrates optimal endosomal escape,resulting in the best knockdown of the target gene relative to thecontrol.

Example 48. Formulation of an ABC Conjugate with Nanoparticles

An exemplary ABC conjugate is packaged into self-assembled nanoparticlesusing cyclodextrin polymers (10 kDa) and an excess of non-conjugatedsiRNAs (ED 40-60 nm, PDI 0.1-0.2). In these particles, the exemplary ABCconjugate maintains its ability to interact with the antibody target.The stability and target binding competency of the particles incirculation in vivo is regulated through modifications of the packagingsiRNAs.

Nanoparticle Formation

Nanoparticles are prepared at a final siRNA concentration of 1.6 mg/mL.siRNA containing CY5-siRNA at a ratio of 1:20 is first diluted to 2×final concentration in water. Cyclodextrin polymer (CDP) is diluted to2× final concentration necessary to achieve a nitrogen to phosphorusratio (N:P) of 3:1 in 10 mM phosphate buffer at neutral pH. CDP is addedquickly to siRNA and is further mixed by pipetting. Particles areincubated for at least 15 minutes before dosing or analysis.

In vitro EGFR binding

Nanoparticles containing various amount of the exemplary ABC conjugateare diluted into Fetal calf serum to a final concentration of 10 nM andare incubated for 1 h at RT with Protein G Dynabeads (Thermofisher)loaded with 150 nM of a purified EGFR-Fc protein (Sino Biological).Beads are washed twice with PBS containing 0.01% Tween 20 and 0.05% BSAbefore bead-bound nanoparticles are disrupted with water containing0.01% Tween 20 and 100 ug/ml heparin. The amount of CY5-siRNA containedin the input, unbound fraction, washes and bead eluate is quantified byfluorescence using a TECAN Infinite M200 Pro (Excitation 635 nm;Emission 675 nm).

CY5-ASC Plasma Quantification

Quantification of nanoparticles in mouse plasma is performed asillustrated in Example 43. The CY5-siRNAs bound to EGFR beads arereleased by using heparin to compete the electrostatic interactionsbetween CDP and siRNAs.

While preferred embodiments of the present disclosure have been shownand described herein, it will be obvious to those skilled in the artthat such embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the disclosure. It should beunderstood that various alternatives to the embodiments of thedisclosure described herein may be employed in practicing thedisclosure. It is intended that the following claims define the scope ofthe disclosure and that methods and structures within the scope of theseclaims and their equivalents be covered thereby.

What is claimed is:
 1. A molecule of Formula (I):A-X—B—Y—C   Formula I wherein, A is an antibody or its binding fragmentthereof; B consists of a double-stranded siRNA consisting of a passengerstrand and a guide strand; C consists of a polymer; and X and Y are eachindependently a linker selected from the group consisting of aheterobifunctional linker, a homobifunctional linker, a maleimide group,a dipeptide moiety, a benzoic acid group or derivatives thereof, a C₁-C₆alkyl group, and a combination thereof; wherein: the double-strandedsiRNA comprises at least one 2′ modified nucleotide, at least onemodified internucleotide linkage, or at least one inverted abasicmoiety; A and C are attached in the same strand, but not attached to Bat the same terminus; A-X and Y—C are conjugated to the passengerstrand; and the molecule has a higher target tumor tissue uptake or ahigher plasma stability compared to an antibody-polymer-nucleic acidconfiguration or an antibody-nucleic acid configuration.
 2. The moleculeof claim 1, wherein the guide strand hybridizes to a target region of agene selected from the group consisting of KRAS, EGFR, AR, CTNNB1,PIK3CA, PIK3CB, MYC, and HPRT1.
 3. The molecule of claim 1, wherein theat least one 2′ modified nucleotide comprises 2′-O-methyl,2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy,2′-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido(2′-O-NMA) modified nucleotide.
 4. The molecule of claim 1, wherein theat least one 2′ modified nucleotide comprises locked nucleic acid (LNA)or ethylene nucleic acid (ENA).
 5. The molecule of claim 1, wherein theat least one modified internucleotide linkage comprises aphosphorothioate linkage or a phosphorodithioate linkage.
 6. Themolecule of claim 1, wherein the at least one inverted abasic moiety isat at least one terminus.
 7. The molecule of claim 1, wherein the guidestrand comprises a sequence having at least 80% sequence identity to SEQID NOs: 16-75, 452-1955, 1956-1962, 1967-2002, 2013-2032, 2082-2109, or2117.
 8. The molecule of claim 1, wherein the antibody or bindingfragment thereof comprises a humanized antibody or binding fragmentthereof, chimeric antibody or binding fragment thereof, monoclonalantibody or binding fragment thereof, monovalent Fab′, divalent Fab2,single-chain variable fragment (scFv), diabody, minibody, nanobody,single-domain antibody (sdAb), or camelid antibody or binding fragmentthereof.
 9. The molecule of claim 1, wherein the antibody or bindingfragment thereof is an anti-transferrin receptor antibody or bindingfragment thereof.
 10. The molecule of claim 1, wherein C is polyethyleneglycol.
 11. The molecule of claim 10, wherein C has a molecular weightfrom about 1000 Da to about 5000 Da.
 12. The molecule of claim 10,wherein C has a molecular weight of about 1000 Da, 2000 Da, or 5000 Da.13. The molecule of claim 1, wherein A-X is conjugated to the 5′ end ofthe passenger strand and Y—C is conjugated to the 3′ end of thepassenger strand.
 14. The molecule of claim 1, wherein Y—C is conjugatedto the 5′ end of the passenger strand and A-X is conjugated to the 3′end of the passenger strand.
 15. A pharmaceutical composition comprisinga molecule of claim 1 and a pharmaceutically acceptable excipient. 16.The pharmaceutical composition of claim 15, wherein the pharmaceuticalcomposition is formulated for the treatment of a solid tumor.
 17. Thepharmaceutical composition of claim 15, wherein the pharmaceuticalcomposition is formulated for the treatment of breast cancer, lungcancer, ovarian cancer, prostate cancer, or skin cancer.